Volume 93  ·   Issue 04  ·  2013

Fabry disease (MIM301500), also known as "Anderson-Fabry disease" (AFD), was first reported by two dermatologists William Anderson (Germany) and Johannes Fabry (England) in 1898, hence the name. The disease is a rare lysosomal storage disease (LSD) inherited by X-associated sex. Its pathogenesis is related to the mutation of α-galactosidase A (α-gal A, a lysosomal enzyme) gene in Xq22. At present, more than 580 mutations have been reported from various ethnic groups^{[1,2,3,4,5,6,7,8,9]}。 The mutation of α-Gal A gene leads to partial or complete loss of the activity of the enzyme, resulting in its metabolic substrate trihexosoylsphingolipid (GL3) and related glycosphingolipid accumulation in various organs and tissues of human body, such as heart, kidney, pancreas, skin, nerves, lungs, etc., and finally causing a series of organ lesions. Since birth, metabolites such as GL3 are deposited in various tissues and organs. Clinical symptoms often appear in childhood to adolescence, and gradually aggravate with the progression of the disease. Many patients, especially male patients, often die of severe renal failure or cardiovascular and cerebrovascular complications in young and middle age. The average survival time of male patients is 20 years shorter than that of healthy people, while the average survival time of female patients is about 10 years shorter^[1,2]Its diagnosis is mainly confirmed by pathological examination, enzymatic assay and α-gal A gene detection on the basis of clinical manifestations.

To study the re-epithelialization of dog′s prostate urethra after two-micron laser resection of the prostate (Tm LRP) and the role of CK34 (CK34βE12) -positive cells in this process.

Tm LRP were performed in 15 elder male dogs and wound specimens harvested at Days 3, 7 and 14 respectively.Immunohistochemical staining was performed to determine the expression levels of CK34 and prostate specific antigen (PSA) in prostatic urethra urothelial cells.

There was no CK34-positive cells in surgical wounds after 3 days while a cluster of CK34-positive cells were found to cover surgical wounds after 7 days,and these cells attached to residual prostate tissues.At Day 14 post-operation,positive expression of CK34 was found in basal cells of newborn urothelium.

Prostate tissues are the important organizations for re-epithelialization of prostatic urethra after benign prostate hyperplasia surgery,and CK34-positive basal cells play an important role in this process.

To test the hypothesis that epithelial-mesenchymal transition(EMT)of prostate cancer is most likely to occur in cancer stem cells(CSC).

The isolation of CSC from LNCaP cell line was performed by flow cytometry based on side-population(SP)phenotype.After SP sorting, LNCaP/SP and LNCaP/NSP were used for further transfection of hypoxia-inducible factor-1α(HIF-1α).Subsequently, EMT-associated proteins were detected by Western blotting.And the assays of Transwell and methyl thiazolyl tetrazolium(MTT)were used to compare invasive and proliferative potency between LNCaP/SP and LNCaP/NSP after HIF-1αinduction.Eventually, xenograft experiments were performed with LNCaP/HIF-1α/SP and LNCaP/HIF-1α/NSP cells for further analysis of in vivo tumorigenesis and distant metastasis.

Through HIF-1α-induced EMT, LNCaP/HIF-1α/SP exhibited such remarkable EMT characteristics as a positive expression of epithelial markers(E-cadherin and CK18)and a negative expression of mesenchymal markers(vimentin, N-cadherin, fibronectin, cathepsin D, MMP-2 and uPAR).And LNCaP/HIF-1α/NSP underwent partial EMT with an abnormal expression of some mesenchymal proteins(vimentin and cathepsin D)and loss of epithelial protein(CK18)despite reservation of another important epithelial marker(E-cadherin).Further Transwell and MTT assays indicated that LNCaP/HIF-1α/SP exhibited stronger in vitro invasive and proliferative potency than LNCaP/HIF-1α/NSP cells.In animal models, the volume of subcutaneous tumor by LNCaP/HIF-1α/SP cells was much greater than that by LNCaP/HIF-1α/NSP counterparts((1008±230)vs(288±145)mm³, P<0.01).Moreover, LNCaP/HIF-1α/SP cells also had a significantly higher rate of subcutaneous tumor incidence(80% vs 53%, P<0.05)and bone metastasis(40% vs 0, P<0.01)as compared with LNCaP/HIF-1α/NSP counterparts.

As the main target cells of prostatic EMT, CSCs may develop a more malignant phenotype after EMT.

To employ enzyme-linked immunosorbent assay (ELISA) to measure the serum level of thrombospondin-1 (TSP-1) and analyze its diagnostic value for prostate cancer.

The serum levels of TSP-1 were measured by human TSP-1 ELISA kit in 50 patients with organ-confined prostate cancer (n=22) and non-organ-confined prostate cancer (n=28).And the subjects of benign prostatic hyperplasia (BPH,n=20) and healthy controls (n=16) were selected.

The average serum concentration of TSP-1 was (200±49) μg/L in prostate cancer group, (281±53) μg/L in BPH group and (323±56) μg/L in healthy control group.There were significant inter-group differences in the serum levels of TSP-1 (both P<0.05).The average serum concentration of TSP-1 was (216±34) μg/L in organ-confined prostate cancer (including stages Ⅰ and Ⅱ) and (188±49) μg/L in non-organ-confined prostate cancer (including stages Ⅲ and Ⅳ) respectively (P=0.030).The level of TSP-1 was also correlated with Gleason score (r=-0.32,P=0.023).However,the relationship between TSP-1 levels and lymph node metastasis remained elusive (P=0.189).The diagnostic sensitivity and specificity of TSP-1 and prostate specific antigen (PSA) for prostate cancer were 72%,90% and 64%,70% respectively (both P<0.05).The area under the receiver operating characteristic curve (ROC) of TSP-1 and PSA were 0.886 and 0.719 respectively (P=0.028).

As a relatively ideal predictor of prostate cancer,the serum concentration of TSP-1 can not only distinguish prostate cancer from BPH,but also correlate with tumor stage and Gleason grade.

To construct a classification and regression tree (CART) to predict the occurrences of bone metastases in patients with newly diagnosed prostate cancer so as to reduce unnecessary bone scans.

CART analyses were performed in 501 subjects from 2005 to 2011 of Fudan University Shanghai Cancer Center to establish Fudan CART model and externally validate Briganti′s CART model.The both models were compared with regards to the area under the curve (AUC) and their clinical values.

The rate of bone metastasis was 27.5% (138/501).The predictive accuracy of Fudan CART model,Briganti′s CART model and skeleton-related events (SRE) model was 0.813,0.691 and 0.645 respectively.There were statistically significant differences (P<0.05).Fudan CART model had a lower missed diagnosis and an over-examination rate of bone scan within the probability threshold (Pt) range of 24.2% to 36.8%.

With a higher predictive value,Fudan CART model may be employed to reduce the unnecessary bone scans for Chinese patients with newly diagnosed prostate cancer.

To compare body composition at birth in the appropriate-for-gestational-age infants of women with gestational diabetes mellitus (GDM) and normal glucose tolerance and determine the influencing factors of body composition in infants of women with GDM and normal glucose tolerance.

A study was conducted on 160 appropriate-for-gestational-age infants (90 males and 70 females) of women with gestational diabetes mellitus (GDM group) and 284 appropriate-for-gestational-age infants (139 males and 145 females) of women with normal glucose tolerance (control group).Anthropometric measurements were obtained within 24 to 48 hours of birth.Multiple stepwise regression was used to determine the correlating factors of fat mass,percent of body fat and fat free mass mass.

There were no significant difference in gestational age,birth weight,length,body mass index,circumferences of head,chest and upper arm,biceps,abdominal superficial skin fold between two groups (all P>0.05),but GDM group was characterized by higher skin folds of triceps and subscapular and flank versus control group (all P=0.000).GDM group had greater fat mass but decreased fat free mass versus control group ( (585±59) vs (480±74) g,17.8%±0.8% vs 14.7%±1.9%, (2685±127) vs (2784±109) g,all P=0.000).Stepwise regression showed that maternal fasting glucose level of oral glucose tolerance test and pregravid body mass index correlated with fat mass and percent of body fat.Fasting glucose level had the strongest correlation with fat mass and percent of body fat (P=0.004,0.006).Gestational age and maternal height correlated with fat free mass in GDM group (P=0.040,0.013).On the other hand,maternal weight gain correlated with fat mass (P=0.015),fasting glucose level and maternal prepartal weight were correlated with percent of body fat (P=0.002,0.043) and pregravid body mass index had correlation with fat free mass in control group (P=0.004).

The appropriate-for-gestational-age infants of women with GDM have increased fat mass and percent of body fat,but decreased fat free mass.Maternal fasting glucose level of oral glucose tolerance test,pregravid body mass index,weight gain and maternal prepartal weight are influencing factors of body composition in neonates.

To analyze the clinical features of fungemia caused by Pichia ohmeri (P.ohmeri) in neonate intensive care unit and explore its molecular biological characteristics so as to improve its diagnosis and treatment level.

The clinical data of 6 infected infants were retrospectively analyzed.The strains obtained from them were identified and homological analysis was performed through randomly amplified polymorphic assay to explore the epidemiological characteristics of this nosocomial infection.

Before the isolation of P.ohmeri,they received intravenous antibacterial therapy for 13—45 days.Among them,4 received mechanical ventilation and 5 had a peripheral insertion of central venous catheters.Five infants were healed after a therapy of caspofungin for 15—30 days.One neonate recurred after a 30-day administration of fluconazole.The strain was identified and confirmed as P.ohmeri.RAPD genotyping results showed that all 6 strains were from the same clone.No similar cases occurred after positive control measures despite a negative epidemiological sampling.

P.ohmeri may cause premature infant fungemia and lead to its spread in hospital.Hospital infection control is a key point.And caspofungin is both safe and effective in the therapy of neonate fungemia.

To explore the related pathogenesis of degenerative aortic valvular disease by observing the histopathological changes of aortic valves from patients with aortic degenerative stenosis and compare the results with those controls with normal aortic valves.

Between May 2009 and May 2010, 22 cases of degenerative calcified aortic valves from patients with aortic valve stenosis undergoing aortic valve replacement (14 males, 8 females, mean age: (66±6) years) and 6 cases of normal aortic valves from those with dissection undergoing Bentall operation (4 males, 2 females, mean age: (43±5)years)were collected.The results of hematoxylin and eosin staining and immunohistochemical examinations were used to observe the histological features of degenerative aortic valves and elucidate the related pathogenesis of degenerative aortic valvuar disease.

Degenerative aortic valve leaflets became thickened.Calcification appeared in aortic side of valve leaflets.Inflammatory infiltrate, angiogenesis, cholesterol crystals, foamy cell aggregation, diffuse and nodular calcification could be seen in subendocardial space of degenerative aortic valve leaflets.No expression of Osterix (OSX)or nuclear factor of activated T-cells 1 (NFATc1)was observed in normal valves.In contrast, the expressions of OSX and NFATc1 showed nuclear immunostaining in degenerative aortic valves.Immunohistochemical staining was graded from 0 to 3.And the expression of OSX was present in 1 (4.5%), 1 (4.5%), 8 (36.4%)and 12 cases (54.5%)respectively in calcified areas, that of OSX in 4 (18.2%), 6 (27.3%), 7 (31.8%)and 5 cases (22.7%)respectively in non-calcified areas, that of NFATc1 in 1 (4.5%), 1 (4.5%), 8 (36.4%)and 12 cases (54.5%)respectively in calcified areas, that of NFATc1 in 4 (18.2%), 6 (27.3%), 8 (36.4%)and 4 cases (18.2%)respectively in non-calcified areas.The expressions of OSX and NFATc1 in calcified areas were higher than those in non-calcified areas (χ²=8.320, P=0.040 andχ²=9.371, P=0.025 respectively).

Unlike in normal valves, inflammatory infiltrate, lipid deposition, angiogenesis and bone regulatory factors appear in degenerative aortic valves.And inflammatory infiltrate, lipid deposition, angiogenesis and ossification may be involved in the degenerative calcified aortic stenosis.

To explore the spectrum of typical symptoms associated with colorectal cancer(CRC)and their influence in timely diagnosis.

A total of 400 cases were randomly collected from 1798 CRC patients treated at our hospital from January 2000 to December 2010.A retrospective chart review was undertaken for all identified cases.Besides demographics and tumor features, TNM stage was obtained from medical records and pathological forms.Other relevant data were as follows：(1)initial symptom complex, (2)presenting symptom or symptom complex, (3)patient duration：period from initial symptoms to consulting a doctor, (4)hospitalization duration：period from initial seeing a doctor to a confirmed CRC case, (5)diagnostic duration：patient duration plus hospitalization duration, (6)rate of misdiagnosis：CRC subjects being diagnosed and treated as other diseases for at least 30 days was identified as misdiagnosis.χ²tests were used to compare the differences of rates of kinds of symptoms and early diagnosis.

Among 400 study subjects, the frequencies of initial symptoms in a decreasing order were change in bowl habits(170, 42.5%), abdominal pain and/or discomfort(157, 39.3%), rectal bleeding(147, 36.8%)and anorectal stimulating symptoms(69, 17.3%).And 176 cases(44.0%)presented multiple symptoms.Short-term health care seeking(≤15 d)after initial symptoms was 40.7%(159/391)within all cases, but only 25.3%(42/166)cases with symptoms of change in bowel habits saw a doctor.The rate was significantly lower than that of other cases(117/225, 52.0%, P=0.000)without this symptom.An onset of symptom complex did not lead to short-term health seeking compare to individual symptoms.Above 50% cases were already in late stage(TNM stage later thanⅡb)as any of the above initial symptoms appeared.No differences of pathology and differentiate of tumors were found among cases with different initial symptoms(P>0.05).Further analyzed the consulting symptoms：rectal bleeding 179 frequency(44.8%), abdominal pain or discomfort 159 frequency(39.8%), change in bowl habits 155 frequency(38.8%), and Anal rectum stimulating symptoms 69 frequency(17.3%).50.8%(203 cases)consulted a doctor with multiple symptoms.59.9%(236/394)cases were of short term hospitalization duration(≤15 d).However, only 52.5%(83/158)in cases whose consulting symptoms included abdominal pain or discomfort, lower than that in other cases without these symptoms(64.8%, 153/236)(P<0.05).High rate of misdiagnosis(50.0%, 10/20)was found in Melena consulter than others(26.8%, 101/377, P<0.05).

Change in bowel habits, rectal bleeding and abdominal pain are the most common symptoms in CRC.And consulting and initial symptoms are similar.Diagnostic and hospitalization delays are seen in half of CRC cases.

To explore the liver disease spectrum in patients with type 2 diabetes (T2DM)and the risk factors of non-alcoholic fatty liver disease (NAFLD).

From September 2009 to October 2011, 1069 hospitalized patients with T2DM in Department of Endocrinology and Metabolism were involved in the study.The history informations, results of laboratory examination, hepatic ultrasound and hepatic proton magnetic resonance spectum (¹H MRS)of all patients were collected to analysis.

(1)The detectable rate of raised liver enzymes in T2DM patients was 28.7% (307/1069), composed mainly of NAFLD (39.4%, 121/307).After excluding the factors such as alcoholic abuse, viral hepatitis, the detect rate of raised liver enzymes in T2DM patients was 26.9% (185/688). (2)The detectable rate of fatty liver by ultrasound in T2DM patients was 56.7% (500/882), composed mainly of NAFLD (72.6%, 363/500), and the detectable rate of NAFLD was 58.0% (363/626). (3)The detectable rate of fatty liver by hepatic ¹HMRS was 72.8% (227/312), composed mainly of NAFLD (69.6%, 158/227).The detectable rate of NAFLD was 69.6% (158/227). (4)Of the three Methods for diagnosing NAFLD, ¹HMRS had the highest detectable rate, followed by ultrasound, and the hepatic enzymes was the lowest.Set the hepatic ¹HMRS as gold diagnosing standrd of NAFLD, the combination of hepatic enzymes and ultrasound increase the sensitivity.The optional cut-off points of ALT were 19.7 U/L (male, ROC_AUC=0.689, P<0.01)and 17.0 U/L (female, ROC_AUC=0.727, P<0.01). (5)Logistic stepwise regression analysis showed sex, BMI, hemoglobin, fasting C-peptide and uric acid (OR=3.803, 1.195, 1.037, 2.896, 1.011, all P<0.05)were positively correlated with NAFLD, and diabetes duration (OR=0.948, P<0.05)was positively correlated with NAFLD independently.

The detectable rate of fatty liver was high in T2DM which was composed mainly of NAFLD.High abnormal liver enzymes detectable rate indicated that NAFLD with T2DM are prone to NASH.

To explore the characteristics and related risk factors of arterial elasticity in persons with prehypertension, high-normal blood lipid and (or)impaired glucose regulation (impaired fasting glucose and (or) impaired glucose tolerance).

After receiving physical and biochemical examinations, a total of 1238 persons were enrolled.Among them, the etiologies were prehypertension (n=65), high-normal blood lipid (n=156), impaired glucose regulation (n=159), prehypertension and high-normal blood lipid (n=85), prehypertension and impaired glucose regulation (n=77), high-normal blood lipid and impaired glucose regulation (n=55)and prehypertension, high-normal blood lipid and impaired glucose regulation (n=9).Also 332 healthy subjects, 113 hypertensive patients, 150 hyperlipidemics and 37 diabetics were enrolled as controls.Systemic vascular compliance (SVC), systemic vascular resistance (SVR), brachial artery distensibility (BAD)were measured with Dynapulse 200 M (Pulse Metric, Inc., USA).

In persons with prehypertension, SVC was lower than healty group ((1.14±0.20)ml vs (1.26±0.23)ml, P<0.01)and higher than hypertensive group ((1.11±0.18)ml, P=0.011), SVR higher than healty group (157±29)kPa·s·L^-1vs (148±25)kPa·s·L^-1, P=0.012)and lower than hypertensive group ((166±36)kPa·s·L^-1, P<0.01)and BAD lower than healty group (5.93%±1.14% vs 6.50%±1.30%, P<0.01).Among different groups with prehypertension, high-normal blood lipid and (or)impaired glucose regulation, SVC, SVR and BAD had significant differences.As indicated by multiple linear regression analysis, blood pressure was an independent risk factor of arterial elasticity.

Vascular function becomes damaged in prehypertensive stage.As an independent risk factor, blood pressure had more potent effect than lipid and blood glucose.Multiple cardiovascular risk factors with high-normal value may affect vascular function more strongly.

To explore the expression of Notch1 receptor in renal tubular epithelial cells transfected with HBx gene and elucidate its role in cell apoptosis.

The eukaryotic vector pcDNA3.1/myc-HBx containing HBx gene or vector pcDNA3.1/myc-Notch1 containing Notch1 gene was transiently transfected into HK-2 cells and shRNA technique employed for silencing Notch1.HK-2 cells were divided into 7 groups of normal culture,pc DNA3.1/myc,HBx,HBx+pcDNA3.1/myc,HBx+Notch1 gene,HBx+shRNA and HBx+Notch1 shRNA.Real-time polymerase chain reaction (PCR) and Western blotting were used to confirm the expression of HBx and detect the expression of Notch1 receptor.Methyl thiazolyl tetrazolium (MTT) assay was used to observe the proliferation rate of HK-2 cells and flow cytometry to detect the apoptosis of HK-2 cells.

HBx and Notch1 receptor were successfully expressed in HK-2 cells post-transfection.The transfection efficiency of shRNA was 70%.The expression of Notch1 receptor in pcDN A3.1/myc-HBx group was higher than that in the normal culture and pcDNA3.1/myc groups.And the apoptotic ratio was also higher than that of normal culture and pcDNA3.1/myc group.The difference was significant (14.94%±0.32% vs 13.86%±0.18%,13.67%±0.54%,both P<0.05).The apoptotic ratio in the HBx+Notch1 gene group was lower than that in control group (10.10%±0.26% vs 14.79%±0.02%,P<0.05).And the growth inhibition ratio of cells was also lower.But the apoptotic ratio and growth inhibition ratio were both higher in HBx+Notch1 shRNA group than those in HBx+sh RNA group (both P<0.05).

HBx gene is successfully transfected into HK-2 cells.And its over-expression may induce apoptosis of HK-2 cells and growth inhibition by regulating Notch1 signal.

To explore the effects and mechanism on anti-leukemic activity of cytokine inducing killer (CIK)cells with an endogenous expression of interleukin-21 (IL-21).

Mononuclear cells were isolated from peripheral blood and cultured with cytokines to generate CIK cells.IL-21 lentiviral vector was constructed and used to transfect 293T cells.Then the culture supernatant with virus infected CIK cells was identified.Proliferation of CIK cells and their cytotoxic activity against K562 cells were measured by methyl thiazolyl tetrazolium (MTT).The expressions of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), perforin, granzyme A, granzyme B, FasL and NKG2D mRNA were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).Immunophenotypes of CIK cells, IL-21 receptor (IL-21R)and Fas L on the surface of CIK cells, intra-cellular perforin and granzyme B of CIK cells were measured by flow cytometry.And the concentrations of IFN-γand TNF-αin cultured supernatant were measured by enzymeimmunoassay.

By restriction enzyme digestion and sequencing, IL-21 lentiviral vector was identified, after transfecting virus surpernatant into CIK cells, the expression of IL-21 was detected in CIK cells.Compared to control, (1)the total number of cells remained unchanged, but the proportion of cells expressing CD3⁺/CD56⁺phenotype increased from 16.95%±4.70% to 24.60%±2.10%. (2)Cytotoxic activity against K562 cells by CIK cells increased from 23.3%±2.8% to 58.4%±8.3% and stayed at 61.2%±6.2% after 5 days.It was stronger and longer compared to the exogenous effect of IL-21 (from 22.8%±2.8% to 44.6%±8.3%). (3)The expression of IL-21R increased around 2 folds. (4)The mRNA expressions of IFN-γand TNF-αincreased almost 1.5 folds, perforin, granzyme B, FasL rose almost 2 folds, the expressions of granzyme A, TNF-βand NKG2D were similar with those of controls. (5)Detected by flow cytometry, the expression of FasL of CIK cells was higher than that of control (0.56%±0.37% vs 0.06%±0.02%), the expression of perforin increased from 12.23%±2.35% to 25.86%±6.13%, the expression of granzyme B rose from 14.56%±1.36% to 37.58%±2.30%, the concentration of IFN-γin culture supernatant spiked from (23.2±5.6)to (55.3±3.5)ng/L and TNF-αjumped from (5.6±0.6)to (15.6±0.6)μg/L.

CIK cells with an endogenous expression of IL-21 have stronger anti-leukemic activity through an up-regulation of IL-21R, perforin, granzyme B, FasL, IFN-γand TNF-α.Thus IL-21 may potentially enhance the anti-leukemic immunotherapy.