BACKGROUND:Human mesenchymal stem cells (hMSCs) become aging and even die after several passages. Some investigations have shown that telomere has a close correlation with life span of the cells. Whether the ectopic expression of human telomerase reverse transcriptase (hTERT) could induce the activity of the telomerase, maintain the length of telomere, and finally prolong the life cycle of MSCs without losing their multipotent differentiation capacity is still uncertain.OBJECTIVE: To observe the influence of the ectopic expression of hTERT on the telomerase activity and cell life cycles of hMSCs.DESIGN: Repetitive measurement trails.SETTING: Research Center of Stem Cell, Zhengzhou University Medical College.MATERIALS: The experiment was conducted in the Research Center of Stem Cell, Zhengzhou University Medical College from October 2003 to December 2005. hMSCs were obtained from 20 healthy donators from the Department of Pediatric Surgery and Outpatient, the Third and First Affiliated Hospitals of Zhengzhou University. Enhanced green fluorescent protein plasmid (pEGFP-C1) and pEGFP-hTERT were provided by Dr. Chantal Autexier of Canada. DH5α strain provided by Dr. Hou Wei-hong, the Key Molecular Medical Laboratory of Zhengzhou University Medical College.METHODS.: Under sterile condition, 2 mL bone marrow of sternum of healthy donors were harvested, and prepared after centrifugalization,dilution and passage.① Transfection of pEGFP-hTERT into hMSCs and the screening and amplification of resistance cloning:The 5th passage cells were seeded in a 24-well plate,and transfected by pEGFP-hTERT with lipofectamine method.The cells were divided into four groups including untransfected group,lipofectamine group,pEGFP-C1 group and pEGFP-hTERT group. Resistance cloning screen and amplification was performed by G418. ②hTERT mRNA expression and detection of telomerase activity:RT-PCR and PCR-ELISA were used to detect the hTERT mRNA expressions of the fifth passage hMSCs transfected with pEGFP-hTERT, and pEGFP-C1, the untransfected tenth passage hMSCs and K562 cells (positive control), and the telomerase activity of the fifth and thirtieth passage hMSCs transfected with pEGFP-hTERT,the fifth pEGFP-C1-transfected cells and the tenth passage untransfected cells. ③Karyotype analysis of hTERT-transfected MSCs: Chromosome analysis was performed by conventional Giemsa staining.④Inducement of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification:The transfected MSCs were cultured in a medium containing epidermal growth factor and basic fibroblast growth factor, which could induce the cells differentiate into neuron-like cells. The culture solution was changed every 3 days, and the changes in cell growth condition and morphologic characteristics were observed under an inverted microscope. The microtubule associate protein (MAP2) and neurofliament subunit M (NF-M) were identified by RT-PCR.MAIN OUTCOME MEASURES:①hMSCs transfection with different kathion liposomes and the screening and amplification of resistance cloning; ②hTERT mRNA expressions of each group and detection of telomerase activity; ③Karyotype analysis of pEGFP-hTERT-transfected MSCs; ④ Induction of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification.RESULTS: ①With the decrease of G418 concentration, the cells in the untransfected and lipofectamine groups died, and stably EGFP expressed MSCs were obtained; after G418 screening, there was a cell clone undergone 35 passages and continued to proliferate, whose appearance and growth characteristics were similar to the untransfected MSCs observed under inverted microscope. ②The fifth passage pEGFP-C1-transfected hMSCs and tenth passage untransfected hMSCs remained telomerase-negative, but the K562 and fifth passage hTERT-transfected cells showed positive telomerase activity. ③The telomerase activity of the fifth and thirtieth passage hTERT-transfected cells was positive. ④The hTERT-MSCs at passage 10, 20 and 30 had 23 pairs of chromosomes, and two X chromosomes. So they were still normal diploid with normal chromosome appearance and number. ⑤Many hTERT-transfected MSCs had the typical appearance of neuron-like cells. RT-PCR analysis showed that th expressions of MAP2 and NF-M were increased.CONCLUSION:Ectopic expression of the hTERT gene is found in hMSCs,and can induce the telomerase activity of hMSCs.The ectopic expression of the hTERT gene in hMSCs could extend the life spans of cells and maintain their multipotent differentiation capacity.

Objective:To determine the correlation of BCG concentration in Freund complete adjuvant ( FCA) and severity of arthritis during arthritis establishment in DBA 1/j male mice induced by bovine type Ⅱcollagen.Methods:CIA was induced by the im-munization of DBA1/j mice with bovine type Ⅱcollagen and BCG of various concentrations dissolved in FCA.To ascertain the effects administering the collagen booster,CIA-related features(including body weight,clinical scoring of arthritis),TNF-αin serum and the histopathological changes in the spleen and the joints regions were measured.Results:4 mg/ml BCG induced more serious arthritis than 1 mg/ml in DBA1/j mice after collagen exposure.Conclusion:There is a positive correlation of arthritis severity with BCG.Which indicates that selection of adjuvant with suitable BCG concentration could determine pathological outcome in CIA mouse model .

Objective To observe the clinical value of indocyanine green retention rate at 15 minutes (ICGR15) in the evaluation of hepatic functional reserve. Methods A total of 185 patients with liver disease, including 45 cases of liver failure, 90 cases of cirrhosis (child A, B and C, respectively), 20 cases of acute hepatitis, 30 cases of chronic hepatitis (mild, moderate). Expression levels of ICGR15 were compared between groups. Values of ICGR15, total bilirubin (TBIL), albumin (ALB), blood coagulation time (PT) were compared before treatment and one month after treatment in hepatic failure group. Levels of alanine aminotransferase (ALT), aspertate aminotransferase (AST), TBIL and ICGR15 were compared before treatment and 1 month after treatment in acute hepatitis group. Results Levels of ICGR15 (%) were 56.3±14.7, 28.9±9.6, 22.4±6.8 and 13.7±2.3 in liver failure group, liver cirrhosis group, acute hepatitis group and chronic hepatitis group, which showed a gradual downward trend (F=125.317, P<0.05). Among them, the levels of ICGR15 (%) were 17.3±5.4, 25.7±7.5 and 34.5±7.3 in Child A, B and C groups of liver cirrhosis group, which showed a gradual upward trend (P<0.05). After one month treatment, levels of TBIL, PT and ICGR15 were significantly lower than T helper 17 cells; intima-media thickness before the treatment in liver failure group. The levels of ALT, AST, TBIL and ICGR15 were significantly lower after treatment than those before treatment in acute hepatitis group (P<0.05). Conclusion ICGR15 can reflect hepatic reserved function, which is not affected by the application of albumin and fresh plasma, and makes up the deficiency of PT and ALB detection.

Objective The main purpose of the present study was to reveal the correlation among the CT and p53?p16 protein of non-small cell lung carcinoma(NSCLC).Methods The expression of p53?p16 protein in 52 cases by SABC immunohistochemical technique and the relationship to CT appearance were analysed.Results (1)The experssion of p53 protein of NSCLC was significantly higher than that of their adjacent tissues and their normal lung tissues(?

AIM: To construct a recombinant retrovirus vector carrying hTERT for establishing UCBMSCs with hTERT(hTERT-MSCs) to overcome their limited life span and detecting whether telomerized UCBMSCs line maintained long-term self-renewal and differentiation capacity.METHODS: The whole cDNA was generated by PCR amplifications from the plasmid pEGFP-hTERT-C1.The hTERT segments were subcloned into pLNCX2.The target cells were infected with these retroviral particles.The stably transfected cells were selected by neomycin and expanded life span which were designated hTERT-MSCs was observed.The expression of hTERT in mRNA level was detected by RT-PCR and the telomerase activity was measured by TRAP(PCR)-ELISA assay.The hTERT-MSCs were induced with 5-azacytidine to cardiac muscle cells and the specific marker of myocardiocyte was detected.RESULTS: The constructed plasmids were digested with restriction endonucleases(BglⅡand NotⅠ).Two characteristic segments including 6.1 kb and 3.6 kb were obtained.The hTERT-MSCs expressed hTERT in mRNA level.The telomerase activity of hTERT-MSCs was positive.The growth kinetics of hTERT-MSCs was higher than those in UCBMSCs.The hTERT-MSCs were induced to myocardiocyte.CONCLUSION: The hTERT recombinant retrovirus vector has been successfully constructed.The hTERT gene activates the telomerase and prolongs the life-span of cells.No effect of hTERT gene on some type of differentiation potential of MSCs is present.

A spectrophotometric method for the determination of calcium in milk, hair and urine was developed using metal complezing dye orthocresolphth-alein complexone(OCPC).The organic matter in the tested sample was destroyed by digestion. An aliquot of the digested solution was mixed with color developing reagent, alkaline OCPC, cantaining 8-quinolinol to suppress interference such as magnesium, and the absorbance was measured at 570 nm.The coefficients of variation were 0.87, 3.6 and 1.2% for milk, hair and urine samples; and the analytical recoveries of calcium from milk, hair and urine averaged 100.4, 101.0 and 98.5% respectively. The calcium content of milk and hair determined by this method was closely correlated with that of the ICP-AES method (r =0.994, p

AIM: To develop and perfect the similarity algorithm on chromatographic fingerprints of traditional Chinese medicine(TCM). METHODS: The experimental and simulated data were used to investigate and verify the characteristics on the new and former methods on similarity evaluation for chromatographic fingerprints method. RESULTS: There are many strong points for new similarity algorithm in practice. CONCLUSION: The new method provides more rationality and practicability in TCM quality control and analysis compare to existing methods.

Adrenal tuberculosis is still the main cause of primary adrenal insufficiency (Addison Disease) in China. A case of bilateral adrenal tuberculosis without PAI symptoms was admitted to Department of Urology, Shanxi Provincial People’s Hospital. Pathological report showed adrenal tuberculosis. We present an overview and discuss how to diagnose early adrenal tuberculosis and reduce misdiagnosis rate so as to preserve residual adrenal function to the greatest extent.

ObjectiveTo evaluate the significance of the detection of serum C-reactive protein (CRP) in elderly patients with diabetes mellitus and cerebral infarction.MethodsSeventy-two patients with diabetes mellitus and cerebral infarction (diabetic cerebral infarction group),66 patients with cerebral infarction and without diabetes mellitus (non-diabetic cerebral infarction group) and 60 healthy persons (control group) from October 2008 to January 2011 were selected.The level of serum CRP was detceted.ResultsThe level of CRP was ( 3.81 ± 2.23 ) mg/L in diabetic cerebral infarction group,( 2.48 ± 2.24 ) mg/L in non-diabetic cerebral infarction group and (0.68±0.16) mg/L in control group.The levels of CRP in diabetic cerebral infarction group and non-diabetic cerebral infarction group were significantly higher than that in control group (P ＜ 0.05).The level of CRP in diabetic cerebral infarction group was obviously higher than that in non-diabetic cerebral infarction group(P ＜0.05).CRP abnormal rate was 70.8％(51/72) in diabetic cerebral infarction group and 43.9％ (29/66) in non-diabetic cerebral infarction group,which had statistical significance (P ＜ 0.05).ConcluslonCRP has important predictive value to the occurrence and development of diabetic cerebral infarction.

Objective To identify differentially expressed N-linked glycoproteins between hepatocellular carcinoma ( HCC) and adjacent non-tumorous liver tissues .Methods N-linked glycoproteome was extracted by multi-lectin affinity chromatography comprising concanavalin A (ConA), lentil lectin (LCH), and snowdrop lectin (GNA) and subsequently subjected to two-dimensional electrophoresis ( 2DE ) and mass spectrometry ( MS ) for identification of differential glycoproteins between 10 pairs of HCC and adjacent non-cancer tissue .Western blotting was used to verify different expression of human liver carboxylesterase 1 (hCE1), haptoglobin (HP)and cathepsin D (CD).Invasion potential in vitro was examined after si-RNA mediated CD gene scilencing .Results LC-ESI-MS/MS identified a total of 28 differentially expressed glycoproteins (14 up-regulation and 14 down-regulated).Western blotting detected consistent down-regulation of hCE1 and HP, and up-regulation of pro-cathepsin D (pCD) in HCC.Up-regulation of ConA-binding CD (ConA-CD), however , was verified in HCC only after ConA-CD enrichment by ConA chromatography .Down-regulation of CD expression mediated by CD-siRNA markedly inhibited the in vitro invasive potential of SNU449 and SNU473.Conclusion Dysregulation of HP , hCE1 expression and alteration of glycans linked to CD may play crucial roles in pathogenesis of HCC.