Volume 93  ·   Issue 14  ·  2013

To explore the functions of connective tissue growth factor (CTGF) in the restoration of hair follicles with a mixture of human dermal papilla cells and human hair follicle outer root sheath cells in vitro in nude mice.

Human hair follicle outer root sheath cells (hf ORS) and human hair dermal papilla cells (hDP) were cultured in vitro and mixed in a fixed ratio (hf ORS∶hDP=5∶1).Flow cytometry was used to detect the content of CD200⁺cells in human hair follicle outer root sheath cells.And 8 nude mice were divided randomly into 2 groups according to a random number table and back wounds produced.Group A was transplanted with cell mixture plus 20μg/ L CTGF.Group B was transplanted with cell mixture alone.After 8 weeks of transplantation, the development of hair follicle formation was observed histologically.PCR was used to detect the expression of human specific DNA and mice DNA in transplants.

The portion of CD200⁺cells in cultured hfORS was 19.65%.At 8 weeks after implantation, hair follicle formation could be observed in Group A (268±96) more than Group B (62±20).The difference was statistically significant (P<0.05).And PCR showed that there was human composition in transplant.

CTGF can induce the formation of hair follicle by promoting the interference between hDP and hf ORS.

To construct and display the keratinocyte growth factor (KGF) phage active peptides so as to detect the promoting effects of epidermal cell.

KGF sequences were chosen and their primers were designed.The selected genes of P1,P2 and P4 were obtained by reverse transcription (RT)-PCR.P3 was obtained by direct synthesis.And the KGF genes were subcloned into pComb3 vector.The technique of phage display was employed to display the genes on phage surface.Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the promoting effects of KGF phage active peptides on the proliferation of epidermal cell.Optical density (A) was determined at 570 nm.Immunofluorescent assay was employed to evaluate the cell affinity of KGF phage active peptides.

The four KGF genes were obtained and subcloned into pComb3 vector.The proteins of the KGF genes were expressed on the surface of the pComb3 vector.The MTT data of optical density (A) showed that significant differences existed between the negative control and KGF control (0.293±0.017 vs 0.520±0.043) and KGF phage active peptide groups (0.293±0.017 vs 0.469±0.057, 0.441±0.048, 0.438±0.035, 0.446±0.037)(all P<0.01).The results of immunofluorescent assay indicated that KGF and KGF phage active peptides had excellent cell affinity.

KGF phage active peptides are successfully constructed and displayed and they may promote the proliferation of epidermal cell.

To contrive an effective method of repairing the scar in bilateral faciocervical region.

Between April 2009 and February 2012, 9 patients with large scars on face and neck due to burn and scald were treated.There were 5 cases with face scars and 4 cervical scars.Their average age was 33 years (range:23-48 years).The disease duration was 6 months to 20 years (mean:6.5 years).The scar area was 12 cm×7 cm to 22 cm×26 cm.The soft tissue expanders (600-800 ml in volume) were implanted in delto-pectoral zone in one-stage operation.In two-stage operation, after the resection of cervical scars, the defects were repaired with delto-pectoral perforator flaps.In 5 facial scar cases, skin flap pedicle division was performed at Week 3.After the resection of scars,all wounds were repaired by expansion flap.The donor sites were sutured directly.The area of removed scar and the status of flap blood supply were observed.And the texture of flaps and patient satisfaction score were followed up for 6- 30 months.

Mild congestion of flap occurred postoperatively 1 case.The other flaps survived successfully.The flaps of 2 cases appeared bulky after transposition and flap repair was performed at Month 6.The appearance, texture, and color of flaps were similar to those at the donor sites.And there was an excellent match of flaps and recipient place.The patient satisfaction score was 7.6±2.3.All achieved satisfactory functional and aesthetic outcomes.

The method has many advantages and its clinical application is both safe and effective.

To explore serial reconstruction strategy for severe cervical cicatrix deformity.

A total of 24 cases, Ⅲ or Ⅳ degree cervical contracture deformity, were treated in Shanghai Ninth People′s Hospital from January 2006 to December 2011.There were 18 males and 6 females with an average age of 35.4 years.The etiologies included burns, chemical injuries and scalding.Three evaluation indices of mental cervical angle (MCA) including soft tissue MCA,osseous MCA and dynamic MCA were measured before treatment and during follow-ups.The first-stage treatment was comprised of cervical cicatrix resection, contracture release, lift of dual direction platysma flap, reconstruction of MCA and skin grafting.At Months 3- 6, second-stage treatment was performed, including lower mandible scar resection, correction of lower lip eversion, lower mandible region reconstruction with free (para-) scapular skin flap.After two-stage treatment, the patients underwent periodical re-evaluations for gross appearance, function and measurement of MCA.

Twenty-two patients completing two-stage reconstruction were followed up.Notable improvement of cervical mobilization and contour were achieved.Soft tissue MCA decreased from 130°±34°to 110°±24°, osseous MCA increased from 71°±23°to 95°±19°and dynamic MCA increased from 25°±18°to 80°±26°.

The serial treatment strategy is effective.In comparisons with reconstruction with skin graft only or skin flap only, the strategy possesses many advantages.

To explore the long-term postoperative complications of Warshaw operation for distal pancreatectomy with preservation of spleen.

Retrospectively analyzed the clinical data of distal pancreatectomy on 84 cases between January 2006 and January 2012.Among them, 35 patients underwent Warshaw operation for distal pancreatectomy with preservation of spleen and another 23 had distal pancreatectomy with Kimura operation.All of them were followed up.

All procedures were successful.There was no perioperative mortality.During the follow-up period, 2 patients undergoing Warshaw operation were lost.The median follow-up period was 2.6 years (range:0.75-6.75).Twentysix patients with the recent neuroimaging data showed:5 patients were diagnosed of splenic infarction and the incidence rate was 19.2% (5/26) and 6 with gastric varices and the incidence rate was 23.1% (6/26).But none of them developed gastrointestinal hemorrhage or splenic abscess.

The Warshaw operation for distal pancreatectomy with preservation of spleen may increase the risk of long-term postoperative complications so that its operative indication must be strictly mastered.

To assess the diagnostic values of urinalysis in urinary tract infections.

A total of 526 urine culture specimens were analyzed by UF-1000i urine analyzers and GEB600 urine chemistry analyzer to determine the sensitivity and specificity of individual and combined analysis of white blood cell, bacteria count and nitrite test in the diagnosis of urinary tract infections.And bacterial culture was used as the gold standard.

The sensitivity, specificity, positive predictive value,negative predictive value and accuracy rate of bacterial count were the highest in individual analysis at 84.3%, 90.5%,84.3%, 90.5% and 88.2% respectively.The sensitivity, specificity, positive predictive value and accuracy of combined bacterial count and leukocyte was 83.9%,91.0%,85.4% and 88.2% respectively while the negative predictive value of white blood cell and nitrite was highest (91.3%).Compared with the combined or single analysis, the combination of three analyses had the highest sensitivity, specificity, positive predictive value, negative predictive value and accuracy rate at 85.4%, 92.6%, 84.7%, 93.0%, 90.3% respectively.

The combined urinalysis may be used as a rapid screening indicator of urinary tract infections.

To explore the ABCC8,KCNJ11,and GLUD1 gene mutations of the 11 patients diagnosed as congenital hyperinsulinism (CHI).

A total of 11 CHI children hospitalized in Beijing Children′s Hospital from November 2008 to February 2012 and their parents were chosen as the study subjects.Direct sequencing of PCR-DNA was used to analyze the 39 exons of ABCC8 gene,non-translational region and exon of KCNJ11 gene and 6,7,10,11 and 12 exons of GLUD1 gene.

An P629PfsX17 heterozygous mutation of ABCC8 gene was detected in case 1 and his father, an W288X heterozygous mutation of ABCC8 gene was detected in case 4 and his father, A640V and Q1196X mutations in ABCC8 gene in case 5 whose father only carried the Q1196X mutation.In case 6 and his father,an R269H mutation was found in GLUD1 gene.The genotype of 4 children′s mothers was normal.No mutations were found in other 7 patients and their parents.

The ABCC8 gene mutations are the main pathogenic mechanisms of Chinese children with CHI.In Chinese, P629PfsX17, W288X, A640V and Q1196X heterozygous mutation of ABCC8 gene and R269H heterozygous mutation of GLUD1 gene may lead to CHI.The inheritance mode of the mutations may be paternally or de novo.

To explore the effects of thyroid hormone on islet β cell function among type 2 diabetics with normal thyroid function.

A total of 266 type 2 diabetics with normal thyroid function were recruited.Their clinical, biochemical parameters and thyroid related hormones were measured.And acute insulin response (AIR) was examined by arginine stimulating test.

The serum levels of triiodothyronine (T₃)((1.49±0.03), (1.59±0.03), (1.70±0.04) μmol/L) and free triiodothyronine (FT₃)((4.01±0.08), (4.37±0.09), (4.44±0.07) pmol/L) were significantly different among tertile groups of AIR (both P<0.01) while those of thyroxine (T₄), free thyroxine (FT₄) and thyroid stimulating hormone (TSH) showed no significant differences among tertile groups of AIR (all P>0.05).A significant positive correlation existed between ln AIR and T₃, FT₃ (r=0.303,0.302, both P<0.01).T₄and FT₄were not correlated with ln AIR (both P>0.05).AIR was significantly different among tertile groups of T₃ ((2.38±0.12), (2.64±0.12) and (3.03±0.10) m U/L) and FT₃ ((2.34±0.11), (2.69±0.13), (2.99±0.10) mU/L,P<0.01).AIR were not significantly different among tertile groups of T₄, FT₄and TSH (all P>0.05).Multivariate linear regression demonstrated that T₃and FT₃level were independently associated with AIR (β=0.686, 95% CI 0.289-0.884, P=0.001,β=0.296, 95% CI 0.125-0.467, P=0.01).

Significant positive associations exist between serum T ₃, FT₃and AIR in type 2 diabetics with normal thyroid function.Serum T₃and FT₃may be the independent risk factors of predicting islet beta cell function in type 2 diabetics with normal thyroid function.However, it remains to be determined whether or not normal physiological concentrations of T₃and FT₃are protective factors for islet β cell functions among type 2 diabetics.

To observe the preliminary effects of closing surgery incision with needleless incision and leaving subcutaneous tissue non-sutured in the operations of abdominal tumor.

There were 52 patients (33 males and 19 females) undergoing abdominal tumor operations between April 2012 and September 2012.Abdominal incision was sutured by a new method of leaving subcutaneous tissue non-sutured closing with a needleless incision close.As a control, the clinical data of 68 patients (47 male and 21 females) received similar operations between April 2011 and September 2011.Abdominal incision was sutured by a traditional method of suturing subcutaneous tissue and skin layer-by-layer.The intergroup incidence of poor healing incident was analyzed by χ² test.Risk factors of poor healing were analyzed by Logistic regression analysis.

No significant difference existed between the new method group and the traditional group as to poor healing of incision (11.54% (6/52) vs 5.88% (4/68),P=0.2666).Age (OR=0.918,95% CI:0.224-3.764), gender (OR=1.371,95% CI:0.364-5.163), wound length (OR=2.567,95% CI:0.52-12.665), preoperative diabetes (OR=1.469,95% CI:0.286-7.554), postoperative serum albumin (OR=0.536,95% CI:0.108-2.655), laparoscopic surgery (OR=0.390,95% CI:0.079-1.922) and suturing method (OR=0.479,95% CI:0.128-1.795) were not risk factors of poor healing.The mean operation duration was 15 min shorter in the new method group than that in the traditional method group.There was no occurrence of scar at either side of incision.

The new abdominal incision closing method does not increase the incidence of poor healing.And this timesaving procedure shows potential cosmetic outcomes.

To explore the therapeutic efficacies of inhaled corticosteroids (ICS) plus low-dose theophylline for moderate bronchial asthma.

A total of 280 patients with moderate bronchial asthma at People′s Hospital of Zhengzhou University between January 2011 and December 2011 were recruited and randomized into 2 groups:observation group with inhaled budesonide 400μg/d plus aminophylline tablet (0.1 g, 3 times/day, oral administration and control group with inhaled budesonide 320μg/day plus formoterol 9 μg/day.The course of treatment was around 6 months.The forced expiratory volume in one second% predicted (FEV₁% predicted),interleukin (IL)-4, IL-5 and IgE of peripheral blood were compared before and after treatment.

Before and 6 months after treatment, the values of FEV₁% predicted, IL-4, IL-5 and IgE for the observed group were 68%±6% and 76%±6%, (14.5±4.4) and (7.2±2.6) ng/L, (27.4±6.2) and (24.2±5.9) ng/L, (771±130) ×10³and (592±104) ×10³U/L, respectively, while those for the control group were 66%±8% and 77%±6%, (13.7±4.3) and (7.7±4.0) ng/L, (26.9±5.8) and (24.6±4.8) ng/L, (752±154) and (604±122) ×10³U/L, respectively.There were significant improvements in both groups (all P<0.05).No differences existed between two groups (all P>0.05).

Compared with ICS plus inhaled long-acting β2-agonists (LABA), ICS plus low-dose theophylline shows similar efficacies in the improvement of lung function and the control of airway inflammation for asthmatics.

To explore the value of sentinel lymph nodes (SLN) metastasis status in predicting the presence of residual disease in non-sentinel lymph nodes (nSLN) and the feasibility of avoiding or reducing the scope of axillary lymph node dissection (ALND) for patients with single positive SLN.

A retrospective study was conducted for 2265 patients with invasive breast carcinomas undergoing sentinel lymph nodes biopsy (SLNB) at Shandong Cancer Hospital between November 1999 and December 2011.And 1228 patients with axillary dissection were screened and divided into 5 groups of (-), (1/n), (1/1), (n/N), (n/n)(n≥2, N≥3, N>n) according to the status of SLN metastasis.

The nSLN metastasis rate of SLN (-), (1/n), (1/1), (n/N) and (n/n) groups was 11.8% (73/618), 25.2% (65/258), 49.6% (67/135), 48.4% (60/124) and 65.6% (61/93) respectively.A comparison of SLN (-), (1/n), (1/1), (n/N), and (n/n) groups of nSLN metastasis showed a significant difference (P=0.000).The differences of nSLN metastasis between SLN (-) and other groups (including 1/n,1/1, n/N, n/n group) were significant (P=0.000).This difference was also significant between SLN (1/n) and other positive groups (include 1/1, n/N, n/n group)(P=0.000), but not significant between SLN (1/1), (n/N) and (n/n) groups (P=0.842, 0.017, 0.042 respectively, Chi-square segmentation).No significant difference existed between axillary lymph node metastasis on LevelⅡandⅢof SLN 1/n group and SLN (-) group (P=0.012, 0.570,χ² segmentation).

The status of SLN metastasis is one of influencing factors for the nSLN metastasis of patients with invasive breast cancer.The possibility of non-sentinel lymph node involvement for patients with single SLN metastasis was smaller than that of other SLN-positive patients.It is safe for some SLN 1/n patients to undergo low lymph node dissection.But ALND is not avoided for patients with single positive SLN (SLN 1/n n≥2).Their clinicopathological variables should be also considered.

To explore the significance of urinary smad3 (usmad3) protein level change in diabetic nephropathy (DN) in type 2 diabetes mellitus and examine its relationship with the progression of DN.

From May 2010 to August 2011, a total of 282 patients with type 2 diabetes were selected for the experimental group according to instant urine specimen albumincreatinine ratio (UACR).Another 100 healthy subjects were taken as the control group.Then the diabetics were divided into 3 groups, including 110 with normal albuminuria (NA group), 114 with microalbuminuria (MA group) and 58 with macroalbuminuria (DN group).Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of usmad3.The parameters of age, systolic blood pressure, body mass index (BMI), blood glucose, blood lipids, urinary albumin/creatinine (ACR), estimated glomerular filtration rate (eGFR) and glycosylated hemoglobin were measured.And non-mydriatic fundus camera was used to evaluate retinopathy,the DN group (n=58) was then divided into two groups of those without retinopathy (n=25) and with retinopathy (n=33).

(1) The usmad3 level in type 2 diabetes group was significantly higher than that in the control group (489 (273, 1193) vs 311 (179, 497) ng/mmol Cr (P<0.01). (2) According to UACR, type 2 diabetes group was divided into 3 different groups to compare the relative level differences of usmad3 in different groups: MA group versus NA group, 552 (316, 1338) vs 317 (200, 594), DN group versus NA group, 1035 (503, 3035) vs 317 (200, 594), DN group versus MA group, 1035 (503, 3035) vs 552 (316, 1338), all P<0.01. (3) Pearson correlation test showed the level of usmad3 was significantly correlated with age, SBP, HbA_1c, blood urea nitrogen, creatinine, total cholesterol, low density lipoprotein, eGFR and UACR (r=0.57, P<0.01).And multiple linear regression analysis showed that usmad3 and U ACR were independently correlated (β=0.754, P<0.01). (4) The usmad3 level in DN with retinopathy were significantly higher than that in DN without retinopathy (1905 (806,4303) vs 595 (331, 1183), P<0.01).No significant difference existed in uACR level between DN with retinopathy and DN without retinopathy (P>0.05).

Urinary level of smad3 is significantly elevated in type 2 diabetics and it is significantly associated with ACR.It suggests that usmad3 is a potential marker in the diagnosis of DN and may be used to predict the severity of DN.

To explore the effects of NF-E2-related factor-2 (NRF2) -617C/ A promoter polymorphism on NRF2 expression as well as lipopolysaccharide-induced inflammatory responses in macrophages.

NRF2 -617C/ A promoter fragments were synthesized by chemical method and cloned into a p UC57 vector.The dul-luciferase reporter assay was employed to determine the activity of promoters.Then recombinant adenoviral vectors were constructed and transfected into macrophages.The expression of Nrf2 was examined by Western blotting and reverse transcription (RT)-PCR.The expressions of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) in macrophages after the stimulation of LPS were determined by enzyme-linked immunosorbent assay (ELISA).

The activity of NRF2 -617C promoter-luciferase reporter (FLuc/RLuc activity ratio) was significantly higher than that of NRF2 -617A group (0.584±0.016 vs 0.258±0.018, P<0.05).The NRF2 protein and mRNA levels in -617C group were much higher than those of 617A group (1.123±0.080 vs 0.951±0.057,1.889±0.031 vs 1.647±0.323, both P<0.05).After the stimulation of LPS, the NRF2 protein expression in macrophages significantly increased (0.584±0.016 vs 0.258±0.018, P<0.05).Compared with -617A group, there was a significantly higher expression of NRF2 in -617C group (0.671±0.033 vs 0.751±0.014, P<0.05).Additionally, the productions of IL-6 and IL-10 in -617C group were markedly lower than those in -617A group as well as IL-6/IL-10 (both P<0.05).However, no significant difference existed in the levels of TNF-α between -617C and -617A groups (P>0.05).

The -617C/A promoter polymorphism of NRF2 may influence the NRF2 expression.And it appears to be associated with the LPS-induced inflammatory responses in macrophages.

To explore the effects of histidine kinase gene CHK1 on some biological characteristics of Candida albicans.

The effects of gene mutation strains of Candida albicans such as CHK21, CHK25, CHK26 and CHK27 were observed on its reproductive ability, formation of chlamydospore and germ tube and tolerance of Congo red.

The reproductive ability in CHK gene mutation strains CHK25, CHK26, CHK27, CHK21 was weaker than that of wild strains (6 h:1.36±0.86,1.25±0.84,1.05±0.79,0.90±0.74 vs 1.54±0.89,P=0.000).And CHK21 was the most obvious.The formation of germ tube in CHK gene mutation strains CHK21, CHK25, CHK26 and CHK27 was weaker than that of wild strains (2 h: 5.6%±2.0%,19.5%±6.9%,13.6%±4.8% vs 29.6%±10.5%,P=0.023,0.028,0.029).Under no light, the mean number of chlamydospore in wild and CHK26 strains was 3 and 22 respectively.With light, the mean number was changed to 60 and 80.So the formation ability of chlamydospore in CHK26 was stronger than other strains.CHK21 could not produce chlamydospore under no light.The mutation strain of CH K1 was sensitive to Congo red.

CHK1 affect the reproduction and formation of chlamydospore and hypha and the tolerance to some environmental pressures of Candida albicans.

To detect the changes of Osterix (Osx) mRNA and protein expression in human periodontal ligament cells (HPDLCs) induced by recombinant human bone morphogenetic protein-2 (rhBMP-2), and examine the role of BMP-2 and Osx during the osteogenic differentiation of HPDLCs.

HPDLCs were isolated and cultured in vitro with explant method.Cells at passage 3 were cultured in DMEM with rhBMP-2 at different concentrations (50,100,150,200,250,300,400,and 600μg/L) for different times (2,3, 5, 7, 10, 14 and 21 days).Then the expressions of Osx mRNA and protein were measured by real-time reverse transcription (RT) -PCR and Western blotting respectively.Cells were treated with 10μmol/L SB203580 (p38 inhibitor) to inhibit p38 phosphorylation induced by rhBMP-2.The mineralization nodules formation and the expressions of phosphorylated p38 and Osx mRNA were detected respectively.

During the culture of rhBMP-2, the expression of Osx mRNA significantly increased.Initially Osx protein had a low expression and then increased in a time-dependent manner followed by the production of bone-like nodules in HPDLCs.Under the effect of SB203580, the up-regulation of phosphorylated p38 expression induced by rhBMP-2 was significantly inhibited as well as the expression of Osx (Osx mRNA expression:0.378±0.034 vs 0.134±0.027, Osx protein expression:0.353±0.024 vs 0.155±0.031, both P<0.01).Meanwhile the mineralization nodules formed by HPDLCs induced by rhBMP-2 were fewer and delayed.

BMP-2 has a significant positive regulatory role on the expression of Osx in HPDLCs.And p38 pathway is an important link of this regulatory process.Thus, as an important signaling pathway in osteogenic differentiation of HPDLCs, BMP-2/p38/Osx may be involved in periodontal tissue remodeling.

To establish Gankyrin knocking down 4T1-luc cell model and detect the effects of Gankyrin expression on breast cancer metastasis.

4T1-luc cells carryring sh Gankyrin construct were established by lentivirus infection and antibiotic screening.Western blotting and real-time PCR were used to check the expression levels of Gankyrin.In vivo imaging system was used to monitor the effects of Gankyrin knocked down on cell growth and tumor metastasis after the in situ implantation of Gankyrin knocking down 4T1-luc cells in BALB/c mice.

The cell expression decreased at the protein and mRNA levels.Gankyrin mRNA expression in different sh Gankyrin 4T1-luc cells was respectively 4.9%,25.1% and 69.8% versus the control cells.ShGankyrin #2 4T1-luc cells were chosen for in situ implantation into BAL/c mice because luminescent intensity was consistent with cell numbers.The photon flux of lung metastatic tumor induced by Gankyrin knocking down 4T1-luc cell was 3.02×10⁶, while that of lung metastasis induced by control cells was 10.9×10⁶. The differences between two groups were significant.In pathology, Gankyrin was detected positive in lung metastasis tumors induced by control group.However, Gankyrin was negative in the Gankyrin knockdown group.

Lentivirus infection may be effectively used to establish Gankyrin knocking down 4T1-luc cell model.Because of its involvement in the in vivo pulmonary metastasis of breast cancers, Gankyrin should be a novel target for tumor therapy.