BACKGROUND: Excessive apoptosis of ovary granulosa cell is a dominant cause of premature ovarian failure and bcl-2 gene is able to inhibit cell apoptosis. But studies demonstrate that,the guanine and cytosine (GC) content reaches 60% in the rat bcl-2 gene sequence. This gene cannot be amplified using routine polymerase chain reaction method. OBJECTIVE: To clone and identify the bcl-2 gene riched GC. DESIGN,TIME AND SETTING: Open experiment was finished in the Laboratory of Medicine and Molecular Biology,Life Science School of Sun Yat-sen University from May to December in 2007. MATERIALS: Wistar rats were purchased from Experimental Animal Center of Sun Yat-sen University. Ecoli DH5?was preserved by Laboratory of Medicine and Molecular Biology,Life Science School of Sun Yat-sen University; pMD18-T vector was purchased from Takara Biotechnology (Dalian) Co.,Ltd. METHODS: The bcl-2-cDNA,in which GC accounted for 60.6%,was obtained by modified reverse transcription-polymerase chain reaction from kidney tissue of Wistar rats,and was cloned into vector-pMD18-T. Characterizations and sequencing of the pMD18-T-bcl-2 were carried out by polymerase chain reaction screening of individual bacterial colonies. MAIN OUTCOME MEASURES: Cloning and purification of bcl-2 gene cDNA; results after connecting bcl-2 gene cDNA to pMD18-T vector and transducting Ecoli DH5?,identification of positive clone and results of sequencing. RESULTS: The bcl-2 gene was identified by the clone and DNA sequencing. DNA sequence analysis was consistent with Genebank sequence,with a 99% homology. CONCLUSION: The gene riched GC is difficult to be amplified,bcl-2-cDNA can be cloned and constructed into cloning vector pMD18-T successfully by the efficient technique for other genes riched GC.

AIM:So far,endostatin is found playing the strongest effect to inhibit endothelial cells.Because endostatin protein is instable,it is difficult to prepare and apply.Gene therapy is used to assist endostatin to inhibit angiogenesis.This study constructed recombinant adenovirus of human endostatin gene using AdEasy-1 system to investigate the feasibility of expressing endostatin protein in ECV304 cells(human umbilical vein endothelial cells).METHODS:The experiment was performed at College of Life Science,Sun Yat-set University from March to October 2006.Endostatin was amplified from Pshuttle-Endostatin plasmid with PCR and subcloned into the pAdTrack-CMV shuttle vector after sequencing and identification.The resultant plasmid was co-transduced into E.coli BJ 5183 cells with pAdEasy-1 plasmid for homologous recombination.The recombinant plasmid was detected by restriction analysis and DNA sequence analysis.The linearized recombinant plasmid was transfected into AAV 293 cells with LipofectAMINE2000.The recombinant adenovirus was detected by GFP,PCR and restriction analysis.The virus was used to infect ECV304,and the expression of endostatin in vitro was detected by the techniques of RT-PCR and Western blot.RESULTS:The positive clones of the recombinants were constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis.AAV293 cells were transfected.The titer of viral liquid was 2.06?1010 pfu/mL.The transcription of endostatin mRNA in ECV304 cells was checked by RT-PCR and endostatin protein could be detected in the culture by Western blot analysis.CONCLUSION:The recombinant adenoviral vector pAd-Endo carrying human endostatin is successfully constructed,and effective expression is found in ECV304 cells in vitro.

0.05). Conclusions Highly purified MSCs can be obtained by Percoll density gradient centrifugation combined with adherent method. CFSE can obtain high labeling efficiency for a short time, and the labeled MSCs maintained the same proliferation ability as non-labeled MSCs. However, along with passage of time, the fluorescence intensity and labeling efficiency decreased.

Aim To investigate the effect of cisplatin(CDDP) on toxicity and apoptosis of human luteinized granulose cells.Method Human luteinized granulose cells were prepared from oocyte retrieval of fertilization procedure in vitro and treated with various concentrations of cisplatin(0,0.5,1.0,2.5,5.0 mg?L-1).The inhibitory rate of cells was examined by MTT assay.The change of nucleolus stained with Hochest 33258 was observed under fluorescent microscope,and bcl-2 and bax protein expressions were detected by flow cytometry(FCM).Results The results showed that CDDP inhibited the growth of ovarian granulose cells at the concentration of no less than 1 mg?L-1,in which Hochest 33258 staining demonstrated cell apoptosis.The expression of bax protein increased as compared with that of the control(P

Objective To clone the full-length cDNA of rat B cell lymphoma-2(bcl-2) gene,then construct and identify the cytomegavirus-mediated lentiviral expression vector of bcl-2 gene,and assess the gene expression in 293T cell,which is a human embryonic kidney cell line.Methods The full-length bcl-2-cDNA fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR) from the kidney tissue of a Wistar rat.The double-stranded oligonucleotides(dsOligoe) were then cloned into the pMD18-T plasmid.After confirmation of a correct construction by sequencing,the positive clone was subcloned into pGC-FU vector with enhanced green fluorescent protein(EGFP),and then transformed into DH5a competent cells.The restricted endonuclease and T4 DNA ligase were used to construct the lentiviral expression vector plasmid pGC-FU-bcl-2 which,combined with the lentiviral packing materials(pHelper 1.0,pHelper 2.0),was then transfected into 293T cell line to form the recombinant lentivirus pGC-FU-bcl-2,and it was used to transfect the 293T cells.The expression of pGC-FU-bcl-2 was further verified by detecting EGFP and bcl-2.Results 1) It was verified by DNA sequencing that the sequence of rat bcl-2 gene was consistent with reported sequence in GenBank.2) The bcl-2 gene was successfully combined in pGC-FU-bcl-2 recombinant plasmid which could be transfected into human embryonic kidney cells.3) The recombinant virus pGC-FU-bcl-2 could be obtained from the 293T cells by co-transfection of pGC-FU-bcl-2 and packing plasmids.4) Targeting gene could be cloned into 293T cells by the recombinant lentivirus with steady expression.The fluorescent protein could be observed under microscope and the expression of bcl-2 protein was detected by Western blotting.Conclusions The lentiviral expression vector containing EGFP and bcl-2 gene has been successfully constructed,with which the transfected 293 T cells can lead to a steady expression of bcl-2 protein.The present study provides a basis for the further study of the function of bcl-2 gene and a potential therapy for the diseases relating to apoptosis.

Objective To reproduce the human endometriosis (EMS) in nude mouse, and to asssess the effects of endostatin in this model. Methods Endometrium tissue was collected from four women undergoing hysterectomy for EMS, and it was transplanted into 30 female BALB/c nude mice by subcutaneous injection (n=20) and intraperitoneal injection (n=10) respectively. The development of endometriotic lesions in nude mice was observed three times every week. Two weeks later, the successful rate in the two groups was estimated. The model mice in subcutaneous injection group were then randomized into treatment group (n=8) and control group (n=7). The mice in treatment group were injected with human endostatin (2mg?kg -1 ?d -1 ), while the mice in control group received an equivalent volume of PBS. All mice were sacrificed 14 days after treatment. Immunohistochemistry was used to determine the expression of vascular endothelial growth factor (VEGF). Results Transplantation was successful in 7 nude mice in abdominal implantation group and 15 nude mice in subcutaneous injection group. The success rate was 75% in subcutaneous injection group and 70% in abdominal implantation group, no significant difference was found between two groups. The level of VEGF in the nude mice treated with endostatin was significantly lower than that in the control group (P

Objective To investigate the anti-apoptotic effect of bone marrow mesenchymal stem cells(MSCs)on chemotherapy-injured ovarian granulosa cells and its mechanism.Methods Granulosa cells were isolated from female Wistar rats and cultured in vitro in media containing chemotherapeutic agents.The experiment consisted of 4 groups:granulosa cells culture group(control group),co-culture group 1 with MSCs/GCs ratio of 1∶1,co-culture group 2 with MSCs/GCs ratio of 1∶5 and co-culture group 3 with MSCs/GCs ratio of 1∶10.Apoptosis was detected by Annexin Ⅴ.The expressions of Bcl-2 and Bax were assessed using Western blotting.The concentrations of vascular endothelial growth factor(VEGF),hepatocyte growth factor(HGF)and insulin-like growth factor-1(IGF-1)in MSCs media were measured by ELISA.Results The drug could induce apoptosis of granulosa cell,and the apoptotic ratio in control group was 35.04%?5.75%,and in the other three co-culture groups was 12.80%?2.42%,13.94%?3.86%,22.31%?5.67%,respectively.There was significant difference in apoptotic ratio between control group and 3 co-culture groups.The expression of Bcl-2 in co-cultured granulosa cells increased significantly compared with that in control group(P

Objective:The characteristics and changes of the three-dimensional micro structure of human placenta in Early-onset severe preeclampsia (EOSP) were quantified, and the relationship of morphologic features and the poor fetal prognosis was explored. Methods:10 patients were recruited in EOSP group, 10 normal blood pressure premature pregnancy were as control group. Ultrathin sections were observed by elec-tron microscope. The Vv,Sv,Nv of plancenta were measured by stereology method. These parameters were compared between the two groups. Results:①In EOSP group, the miorovillus at the bottom of syncytial was sparse and swollen. The mitochondria and endoplasmic reticulum were swollen and dilated. The deposition of lipid granules were increased.②The Vv, Sv of mitochondrial and endoplasmic reticulum in EOSP group were significant augmented than control groups( P<0.01 ). There was no significant difference of Nv in mitochon-drion and endoplasmic reticulum between two groups ( P >0.05). ③The Vv,Sv, Nv of microvilli in EOSP group were smaller than those in control group ( P < 0.01 ). Conclusions. In EOSP, the microstructure of placenta is impaired, thus the synthesis and transportation function of placenta were affected, so fetus could be injured subsequently.

Objective:To investigate the wether vascular endothelial growth factor (VEGF) and endostatin could be used as new serum markers of endometriosis. Methods:Preoperative serum levels of VEGF and endostatin in 82 women with endometriosis(stage Ⅰ-Ⅱ 43 cases and stage Ⅲ-Ⅳ 39 cases) were assayed in duplicate using enzyme linked immunoadsordent assay. Serum levels were compared with levels of 60 healthy controls. Results: Serum VEGF and endostatin levels in endometriosis were significantly higher than that of controls. The levels of VEGF and endostatin of endonmetriosis stages Ⅰ-Ⅱ were significantly lower than those of stage Ⅲ-Ⅳ. The sensitivity, specificity, positive predictive value and negative predictive value of serum VEGF in detecting endometriosis were 0.92,0.78,0.82 and 0.90, respectively; those of enedostatin were 0.95, 0.84, 0.86 and 0.95, respectively. When both factors were used concomitantly, they were 0.98,0.75,0.76 and 0.98, respectively. Conclusion:These results indicates that the balance of angiogenic stimulators and inhibitors may regulate the development and progression of endometriosis and demonstrates that the circulating levels of both VEGF and endostatin maybe useful markers for endometriosis.

Objective To compare the effects between dilation and curettage (D&C) and hysteroscopic electroresection (HE) in the management of endometrial polyps. Methods Entered into the study there were 86 patients with endometrial polyps, 32 of them were treated by D&C (Group D&C) and 54 by HE (Group HE). The operation time, pre- and post- operative complications, and recurrence of 2 groups were compared. Results Of the Group D&C and Group HE, the operation time were (8.5?4.2) min and (9.0?3.1) min, without statistically significant differences (t=0.632, P=0.529), while the recurrence numbers and recurrence time of the 2 groups were 9 (36.0%) and 3 (7.5%) cases, (5.3?3.5) months and (11.2?4.8) months, respectively, with significant differences (?2=6.516, P= 0.011; t=6.058, P=0.000). Conclusions Compared with D&C, HE is characterized by lower recurrence rate and longer recurrence time.