Volume 85  ·   Issue 27  ·  2005

Stem cell transplantation can be traced back to the use of bone marrow cells to treat leukemia in 1950s. Later, with the maturity of peripheral blood cell separation and purification technology and the discovery of cell surface antigen markers, people began to notice that the essential cells of early bone marrow transplantation for leukemia are bone marrow hematopoietic stem cells, that is, bone marrow cell transplantation is actually bone marrow hematopoietic stem cell transplantation.

The research of stem cells is in the ascendant.Because embryonic stem cells involve ethical issues, adult stem cell research has attracted much attention, among which mesenchymal stem cells are the most widely and deeply studied adult stem cells.It has been confirmed that mesenchymal stem cells exist in bone marrow, skin, dermis, fat, muscle, periosteum and other tissues.A large number of experimental studies and some clinical trials have shown that mesenchymal stem cell transplantation has a good prospect for the treatment of hematopoietic recovery, wound healing, bone and cartilage tissue replacement, myocardial and skeletal muscle repair, vascular repair and some neurological diseases [1,2]. Our own research also found that mesenchymal stem cell transplantation can simultaneously promote hematopoietic recovery and wound healing [3].

Vision is one of the most important indicators to guarantee the quality of human life. There are hundreds of millions of visually impaired patients in the world in the darkness, and the stem cell research in recent years has brought dawn for these patients. However, there are still many mysteries in the field of stem cells to be revealed, and the internal molecular regulatory mechanism of stem cell differentiation is still unknown, which poses a challenge to scientists.

Most adult vertebrates have limited regeneration ability, and only some tissues and organs have strong repair and regeneration ability, among which adult stem cells (ASCs) play a key role. 　　

In the field of life science, the advances and breakthroughs in human embryonic stem cells and cloning technology have brought revolutionary influences to medical treatment and health. At the same time, the bioethical problems caused by related research results have attracted much attention. How to better coordinate and solve the ethical debate faced in the process of stem cell research has become a very realistic and urgent problem for people to solve.

Objective to evaluate the therapeutic regimen of levofloxacin. Methods Twelve healthy volunteers were given a single dose of levofloxacin (200, 300 and 500 mg) intravenously to measure the blood and urine drug concentrations, and calculate the pharmacokinetic (PK) parameters. In vitro pharmacodynamic (PD) study was conducted to determine the drug susceptibility parameters [minimum inhibitory concentration (MIC)] of levofloxacin to 823 recent clinical isolates, and compared with other related antimicrobial drugs. According to the above results, the PK/PD parameters of levofloxacin peak concentration (Cmax) /MIC and area under the plasma concentration-time curve (AUC) /MIC were calculated. According to these results, levofloxacin was recommended for the treatment of different pathogenic infections. Results The results of clinical PK study showed that after a single dose of levofloxacin (200, 300 and 500 mg) intravenously, the Cmax was 3.4 mg/L ± 0.8 mg/L, 4.8 mg/L ± 1.4 mg/L and 7.6 mg/L ± 1.1 mg/L, respectively, and the AUC0-∞ was 14.4 mg · h/L ± 2.5 mg · h/L and 21.9 mg · h/L ± 4.5 mg · h/L, respectively In vitro PD study showed that levofloxacin had high antibacterial activity against hemolytic Streptococcus and Moraxella catarrhalis, and had good antibacterial activity against Streptococcus pneumoniae (including penicillin-insensitive strains), Haemophilus influenzae, methicillin-sensitive Staphylococcus aureus, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Pseudomonas aeruginosa and Klebsiella pneumoniae. Methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus were mostly resistant to levofloxacin, while more than half of Escherichia coli were resistant to levofloxacin. 300 mg daily is expected to be effective in the treatment of hemolytic streptococcal infection, 500 mg daily is expected to achieve good curative effect in the treatment of respiratory tract infection caused by Streptococcus pneumoniae and Haemophilus influenzae, while at least 500 mg daily is required in the treatment of respiratory tract and urinary tract infection caused by Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Stenotrophomonas maltophilia which are sensitive to this drug. However, for MRSA and enterococcal infection, PK/PD parameters indicate poor curative effect. Conclusion The dosing regimen of levofloxacin based on PK/PD principle is expected to achieve good clinical efficacy.

Objective to investigate the expression of Thy-1 in lung cancer tissues by immunohistochemical staining, analyze its effect on the prognosis of patients with non-small cell lung cancer, and understand the correlation between the expression of Thy-1 and p53, so as to provide ideas for further research on the expression mechanism of Thy-1 in lung cancer. Methods The expression of Thy-1 in 175 lung tissues including different pathological types was detected by immunohistochemical signal catalytic amplification method in the form of tissue microarray, and the combined detection of Thy-1 and p53 in 91 cases of non-small cell lung cancer was performed. The correlation between the expression of these two proteins was analyzed, and the relationship between Thy-1 and survival and prognosis of patients was analyzed by combining clinical pathological data and follow-up results. Results Thy-1 antibody specifically showed strong positive staining in the nucleus of lung cancer cells of different pathological types, and 89 cases (56.7%) were positive for Thy-1 nucleus staining. The univariate and multivariate analysis of prognosis of 91 cases of non-small cell lung cancer showed that, TNM stage, lymph node metastasis and high expression of Thy-1 in the nucleus were independent factors affecting the prognosis of patients. The stratified study showed that the survival time of Thy-1 nucleus-negative patients was significantly longer than that of positive patients in the group without lymph node metastasis (46.42 months and 38.56 months, respectively, P =0.0207). There was a significant correlation between the expression of Thy-1 and p53 (P<Conclusion the specific high expression of Thy-1 in the nucleus of lung cancer is one of the prognostic factors of non-small cell lung cancer. The abnormal high expression of Thy-1 in the nucleus can be used as a marker for pathological diagnosis of lung cancer.

Objective to detect the differential expression of cyclooxygenase 2 (COX-2) and caspase-3 proteins in the formation of lung squamous cell carcinoma induced by methylcholanthracene and to explore their significance. Methods Lung squamous cell carcinoma of Wistar rats was induced by infusion of lipiodol solution containing methylcholanthracene 10 mg and diethylnitrosamine 0. 01 ml into the left lobe bronchus. The expression of COX-2 and caspase-3 proteins in each stage of lung squamous cell carcinoma was detected by immunohistochemical SP method and the immunohistochemical score (IHS) was calculated. Results 80.0% (64/80) of animal specimens with multiple stages of lesions in the experimental group, among which 14 cases of bronchial membrane epithelial hyperplasia, 25 cases of squamous metaplasia, 35 cases of atypical hyperplasia, 12 cases of carcinoma in situ, 54 cases of invasive carcinoma and 15 cases of metastatic carcinoma were obtained. The weak positive expression of COX-2 in normal bronchial mucosal epithelium was occasionally observed, and the IHS of COX-2 in dysplasia, carcinoma in situ and metastatic carcinoma increased gradually, with highly significant differences (all P<The positive expression of caspase-3 was observed in 8 cases (80.0%) of bronchial mucosal epithelial cells in the control group. The HIS of caspase-3 decreased gradually in the stages of dysplasia, invasive carcinoma and metastatic carcinoma, and the difference was significant (P<0.01, P<0.01, P<There was a significant negative correlation between COX-2 and caspase-3 (χ 2=106.664, spearman's correlation coefficient was-0.678, P<Conclusion The increase of COX-2 protein and the decrease of caspase-3 protein are closely related to the formation of lung cancer, and the interaction between them promotes the occurrence and invasion of lung cancer.

Objective to observe the effects of anesthetic and analgesic drugs, somatostatin analogues and nitrate drugs on the motor function of Odie's sphincter by choledochoscopic manometry, and to observe the antagonistic effects of M-cholinergic receptor blockers and nitrate drugs on morphine. Methods 157 patients with T-tube after biliary tract surgery due to gallstones were randomly divided into anesthetic and analgesic drugs group, somatostatin group, nitrate group, nitrate and morphine combination group and M-cholinergic receptor blockers and morphine combination group. The duodenal pressure, Odie's sphincter basal pressure, systolic amplitude, systolic frequency, systolic interval and common bile duct pressure before and after administration were measured. Results After intramuscular injection of morphine and Qiangtongding, Odie's sphincter basal pressure (SOBP) and systolic amplitude (SOCA) increased, and the systolic frequency increased faster, while the common bile duct pressure (CBDP) increased in the morphine group. There was no obvious change in all indexes after intramuscular injection of Dulengding; After intramuscular injection of trimodol, SOBP and amplitude of contraction were significantly decreased. After injection of statanine at the rate of 250 μ g/h, the amplitude of contraction was increased. After the rate was adjusted to 500 μ g/h, SOBP and amplitude of contraction were significantly decreased compared with before the dose was doubled. After intravenous injection of Shanning, only CBDP was significantly increased. After sublingual administration of xiaoxintong or nitroglycerin, SOBP and amplitude of contraction were significantly decreased. After 20 min administration of pentylene tetranitrate, SOBP decreased. After 10 min administration of morphine, SOBP and amplitude of contraction were significantly decreased and the frequency of contraction was slightly slowed down. After 10 min administration of morphine, SOBP and amplitude of contraction were decreased by intramuscular injection of anisodamine and atropine. After 10 min administration of morphine, SOBP and amplitude of contraction were decreased. Trimodol has inhibitory effect on Odi's sphincter; Dulengding and Shanning had no obvious effect on Odi's sphincter; Stenin excites the Odie sphincter at low doses; Nitrate drugs have inhibitory effect on Odi's sphincter; Nitrates and M-cholinergic receptor blockers can antagonize the agonistic effects of morphine.

Objective to investigate the secretion and synthesis of insulin and glucagon in islets, the secretion of glucagon and insulin under high glucose stimulation and the changes of insulin signal transduction molecules in islets in obese rats with peripheral insulin resistance (IR) induced by high fat diet.Methods Thirty male Wistar rats were randomly divided into obese group fed with high fat diet and control group fed with normal diet, 15 rats in each group for 20 weeks. Dynamic secretion changes of insulin and glucagon in high glucose state were detected by surface perfusion of islet cells; The expression of insulin receptor (IRc) and insulin receptor substrates 1, 2 (IRS-1, IRS-2) in the islets of the two groups was detected by immunohistochemical staining and image analysis. Results (1) The insulin sensitivity index (ISI) of the obese group was significantly lower than that of the control group, and the blood glucagon level and the glucagon level in the islets of the obese group were significantly higher than that of the control group (362 pg/ml ± 58 pg/ml vs 291 pg/ml ± 35 pg/ml, P<0.05; 442 pg/ml ± 56 pg/ml vs 287 pg/ml ± 48 pg/ml, all P<(2) Glucose-stimulated insulin secretion (GSIS) was impaired in the obese group. 16.7 mmol/L glucose significantly inhibited glucagon secretion from islet α cells in the control group, but this inhibitory effect disappeared in the obese group. (3) The expression of IRc, IRS-1 and IRS-2 in the islets of the obese group was 28% and 22% lower than that in the control group, respectively (both P<Conclusion the insulin signal transduction pathway of islet cells in obese rats was impaired by high fat diet, that is, peripheral IR and intrapancreatic IR were present, which may be one of the internal mechanisms of islet cell dysfunction in obese rats.

Objective to investigate the expression of tyrosine protein phosphatase 2 (Shp-2) in chronic myeloid leukemia (CML) cells and its role in malignant proliferation and apoptosis resistance induced by p210bcr/abl. Methods 25 leukemia cells from p210bcr/abl positive CML patients, 8 non-tumor patients' bone marrow and 10 normal subjects' peripheral blood cells were collected as negative controls, K562 and KU812 leukemia cells were used as positive controls for p210bcr/abl, and KG-1 leukemia cells were used as positive controls for Shp-2. The expression of Shp-2 in leukemia cells and normal bone marrow hematopoietic cells was quantitatively analyzed and compared by Western blot technique, and the effects of Shp-2 expression on the proliferation and apoptosis of p210bcr/abl positive leukemia cells were observed after downregulation of Shp-2 and leukemia fusion gene p210bcr/abl by specific inhibitors The ratio of phosphorylated Shp-2/β-actin was 0.91±0.62, 0.16±0.09 and 0.03±0.05, respectively<(2) The apoptosis rate of leukemia cells increased from 4.89% to 38.69% (P<0.01), while S-phase cells decreased from 33.6% to 10.8% (P<(3) After specific inhibition of p210bcr/abl fusion gene expression protein, the phosphorylated Shp-2 protein in leukemia cells decreased significantly, and there were obvious apoptosis and growth inhibition phenomenon.Conclusion (1) The phosphorylated Shp-2 protein is overexpressed in patients with chronic myeloid leukemia cells, and it is closely related to malignant proliferation and apoptosis resistance of leukemia cells. (2) The overexpression of Shp-2 in leukemia cells of patients with bcr/abl fusion gene positive may be caused by the activation of p210bcr/abl protein.

Objective to study the effects of blood glucose and sugar content in skin tissue on wound healing of superficial second degree scald. Methods Ninety-six clean grade SD rats were randomly divided into normal group and STZ-induced immediate-onset diabetes group, and the model of superficial second degree scald with 10% total body surface area (TBSA) was induced. The blood glucose and sugar content in skin or wound tissue were measured before injury, on the first, third, fifth, seventh, tenth and fourteenth days after injury, respectively. Gross and histological dynamic observation of wound healing process; The proliferation cycle of epidermal cells was detected before injury and 3, 7 and 14 days after injury. Results the blood glucose concentration of diabetic rats was significantly higher than that of normal rats (27.28 mmol/L ± 0.80 mmol/L vs 4.65 mmol/L ± 0.14 mmol/L, P<0.01); There was a significant correlation between sugar content and blood glucose (r =0.881, P<0.05), the local glucose content of shallow second degree scald wound in diabetic group was significantly higher than that in normal scald group (14.2 mmol/L ± 3.0 mmol/g vs 4.5 mmol/L ± 0.5 mmol/g, P<Conclusion the increase of sugar content in local wound tissue of diabetic rats is closely related to the change of blood glucose, and the increase of sugar content in local wound is inseparably related to the delay of wound healing. High glucose environment can inhibit the proliferation of epidermal cells in shallow second degree scald wound.

Objective to explore the construction of tissue engineered cartilage using the acellular matrix of allogeneic cartilage particles as scaffold. Methods The acellular matrix of sheep articular cartilage was prepared by multi-step enzymatic method, and the contents of collagen, glucosamine and DNA of the microparticles were measured by gross, light microscope (hematoxylin-eosin, collagen, toluidine blue staining) and scanning electron microscope. The contents of collagen, glucosamine and DNA of the microparticles were measured simultaneously. The allogeneic sheep articular cartilage cells were isolated, expanded in vitro, and mixed with the acellular matrix of cartilage particles were cultured in vitro for 0~35 days. The contents of hydroxyproline, glucosamine, DNA and cell adhesion rate were measured by inverted microscope, scanning and transmission electron microscope. Results The acellular matrix of cartilage particles only contained matrix components, with a diameter of 0.100~0.154 mm. The contents of collagen, glucosamine and DNA were 204.4 ± 3.1 μ g/mg, 18.3 ± 2.0 μ g/mg and 0.042 ± 0.013 μ g/mg, respectively. The contents of collagen, glucosamine and DNA in the complex formed by allogeneic chondrocytes tightly surrounded the acellular matrix of allogeneic cartilage particles were statistically significant (P<0.05) compared with the contents of collagen, glucosamine and DNA in the complex formed by allogeneic chondrocytes on the 7th day and on the 0th day, and reached the peak at the 14th day (collagen, DNA) and the 21st day (glucosamine), respectively, and then maintained at a high level.

Objective to investigate the expression of immune molecules on the surface of adipose derived stem cells (ADSCs) and the function of regulating lymphocyte response in vitro, so as to provide allogeneic seed cell source for tissue engineering.Methods adipose stem cells (3 cases) were isolated from adipose tissue discarded from clinical liposuction, cultured in vitro to the second passage, and the expression of immune molecules HLAI and HLAII was detected by flow cytometry.1×105 ADSC cells/well were stimulated to observe the proliferation of allogeneic lymphocytes, and the lymphocytes were stimulated with phytohemagglutinin (PHA, 2 μ g/ml), and then (1×102, 1×103, 1×104, 1×105) allogeneic adipose stem cells/well were added to the reaction system to observe the proliferation of lymphocytes. In the other group, interleukin-2 (IL-2, 0.5 ng/ml) was added at the same time to detect the reversal of the inhibitory effect of adipose stem cells. The number of "third party" adipose stem cells or adipose stem cells treated with IFN-γ were added to the mixed bidirectional lymphocyte response system for 48 h, respectively, to observe the inhibition of lymphocyte proliferation. Results the adipose stem cells expressed HLA class I molecules, but no positive expression of HLA class II molecules was detected. After 48 h of human IFN-γ stimulation, the expression of HLA I and HLA class II molecules was not significantly increased. Neither allogeneic nor IFN-γ-treated adipose stem cells could stimulate the proliferation of lymphocytes in vitro. Adipose stem cells significantly inhibited the proliferation of lymphocytes stimulated by PHA, and IL-2 could reverse the inhibitory effect of adipose stem cells. Adipose stem cells significantly inhibited the biphasic mixed lymphocyte response, and the inhibitory effect was dependent on the number of adipose stem cells; Conclusion ADSC can inhibit the proliferation of lymphocytes in vitro, and has the ability to regulate the lymphocyte response to a certain extent, and may be a source of allogeneic cells in tissue engineering or cell therapy.

Objective to investigate the transformation of human mesenchymal stem cells (hMSCs) and injured human sweat gland cells (hSGCs) in vitro. Methods hMSCs and hSGCs were isolated and amplified in vitro, and the antigen expression of hMSCs and hSGCs was detected by two-step immunocytochemistry. The cultured hMSCs were continuously labeled with 5 μ mol/L 5-bromo-2-deoxyuridine (BrdU). After 70% of the primary cultured hSGCs were fused, the hMSCs were treated at high temperature at 47℃ for 40 min to induce thermal injury in vitro model, cooled at 37℃ for 1-2 h, and then (1-2) ×105 BrdU-labeled hMSCs were added to co-culture. After 2 weeks, anti-carcinoembryonic antigen (CEA) and anti-BrdU monoclonal antibody were used as primary antibodies, and the co-cultured cells were detected by immunocytochemical double staining method. Results hMSCs and hSGCs showed clonal growth, and hMSCs expressed CD44 and CD105, but did not express CD34 and CEA hSGCs expressed CK7, CK8, CK19 and CEA, and the positive rate of hMSCs labeled with BrdU could reach 90%. After hSGCs were injured by high temperature, the intercellular junction of most cells disappeared. After 2 weeks of co-culture, some cells expressed CEA and BrdU simultaneously, and there was multinuclear phenomenon. Cell staining showed that the number of double-stained cells reached 1% ~5%, and the number of multinucleated cells with different nuclear staining was about 0.01% ~0.05%. Compared with other double-stained cells, the cells of multinucleated cells were wider and flatter. Conclusion hMSCs can transform into hSGCs under injured microenvironment, and the mechanism may be cell differentiation, cell fusion and even nuclear fusion.

Objective to point out the crux of misuse and abuse of statistics from the academic angle, and put forward creatively the "three-type theory of statistics" which can solve the aforementioned problems fundamentally, namely the "phenotype", "prototype" and "standard type" of the problems. Methods to clarify the aforementioned "three-type theory" is the essential of rational application of statistics from both theoretical and practical aspects, and to show that the "three-type theory" is related to each other through examples. Using the "three-type theory" to explain and analyze the experimental design and statistical analysis problems in biomedical research can help people see the essence of things through the phenomenon. Results Through research, it is found that the "three-type" of some problems are the same with each other, the prototype of some problems is the standard type, but the "three-type" of some problems are different from each other. Especially in some multi-factor experimental studies, researchers make the mistake of "incomplete comparison", resulting in the "standard type" corresponding to the "prototype" does not exist at all, This is a problem that needs to be solved by introducing the idea and method of "splitting". Conclusion Once the "three types" of each specific problem are clarified, it is a matter of course to design experiments reasonably and choose statistical analysis methods correctly to deal with specific experimental data. "Three types of statistics theory" can help people not make statistical mistakes in the process of using statistics or at least greatly reduce the chance of misusing statistics, can greatly improve the quality, level and speed of biomedical scientific research, can improve the quality and teaching effect of statistical textbooks, and point out the forward direction for the development of biomedical statistics.

In recent years, the research on embryonic stem cells has been deepened, and the understanding of the biological characteristics of adult stem cells has been further deepened. adult stem cells are a group of undifferentiated (or specialized) cells that exist in differentiated (or specialized) tissues. Besides their strong self-renewal, proliferation and differentiation potential, they also have great plasticity. The discovery of plasticity phenomenon of adult stem cells makes people hopeful for the future application of cell regeneration to treat tissue defects caused by burns, wounds or other diseases.

The International Bioethics Symposium with the theme of "Medical Market, Morality and Traditional Cultural Resources for the 21st Century" was held in the School of Medicine of Shandong University from 27 to 28 June 2005. The conference was jointly sponsored by the School of Medicine of Shandong University, the Asian Governance Research Center of City University of Hong Kong, the International Society of Philosophy and Medical Research (USA) and the Journal of Medicine and Philosophy (USA). More than 20 experts and scholars from the United States, Hong Kong, China and mainland China gathered in Shandong University to discuss hot issues in the field of bioethics and medical care.

The 2005 Academic Annual Meeting of the American Heart Rhythm Society (formerly the North American Pacing and Electrophysiology Society, renamed since 2005) was held in New Orleans, USA from May 4 to 7, 2005. Besides the full exchange and introduction of various clinical advances in cardiac pacing and electrophysiology, there were also many reports on basic research related to clinical heart diseases. The special reports of the four sub-forums are presented as follows.

Case 1: A 40-year-old male suffered from painful urination 1 year ago, obvious at the end of urination, and paroxysmal pain in the lower abdomen. The painful urination was aggravated with macroscopic hematuria in the past 5 days. Cystoscopy showed that the tumor was about 1.5 cm ×1.5 cm at the top of the bladder, with narrow base.

As new insulin sensitizers, pioglitazone and rosiglitazone have been widely used in clinic, and their effects on blood lipids have attracted considerable attention. Previous studies were either retrospective analysis or without strict control, and the conclusions obtained were not persuasive.Goldberg, University of Miami School of Medicine, USA, found that the effect of pioglitazone on blood lipids was significantly better than that of rosiglitazone through multicenter, prospective, randomized, double-blind and parallel-controlled studies.This result was published in Diabetes Care, July 2005 (Diabetes Care, 2005, 28:1547-1554).

Laparoscopic surgery has many short-term advantages, such as less postoperative pain, less wound complications, quicker postoperative recovery, short hospital stay, etc. A study published by Lancet on May 14, 2005 (Lancet, 2005, 365:1718-1726) stated that compared with open surgery, laparoscopic assisted resection has the same short-term efficacy in the treatment of colon cancer, and may produce similar long-term efficacy.

Irritable bowel syndrome (IBS) is a functional bowel disease characterized by abdominal pain/abdominal discomfort with changes in defecation habits. The incidence rate of this disease is high, and the reported in Beijing and Guangzhou in China is 0.82% and 5.67%, respectively [1]. The pathogenesis of this disease is still not completely clear. Studies have shown that serotonin (5-HT) and genetic factors may be related to the pathogenesis of IBS [2]. There are multiple polymorphism sites in human serotonin transporter (SERT) gene, and foreign studies have shown that 5-HTTLPR in the promoter region and variable number tandem repeat (VNTR) polymorphism in the second intron region are related to IBS [3-5]. There are few relevant reports in China, so we conducted a controlled study on the polymorphism of SERT gene in IBS patients and healthy people.

A 55-year-old male was admitted to the Sir Run Run Shaw Hospital affiliated to Zhejiang University in February 2005 because of yellowing skin and white eyes. Physical examination showed that the general condition was acceptable, with obvious yellowing of skin and sclera, flat and soft abdomen, no tenderness, and no subcostal pain in liver and spleen. Before admission, he went to a local hospital. ERCP showed that the intrahepatic and extrahepatic bile ducts were dilated and no stones were found. CA19-9: 1617 U/ml, total bilirubin 142 mg/L, direct bilirubin 98 mg/L; After admission to our hospital, ERCP examination and nasobiliary drainage were performed, ERCP biopsy was negative, and jaundice resolved after 2 weeks of nasobiliary drainage; Complete laparoscopic pancreaticoduodenectomy was performed under general anesthesia on 9 March 2005 after adequate preoperative preparation.