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.

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.

There are so many patients with different liver diseases in our country that the diagnosis and treatment of liver diseases directly relate to the people's healthy level.The requirement of diagnosis and treatment to clinical laboratories can be summarized as: ( 1 ) sensitivity,which means shortening window period;(2) specificity,which means various subtypes and variant can be detected;( 3 )speed,which means finding out the pathogen as soon as possible;(4)accuracy,which means providing physicians with accurate test reports.Detecting techniques,for instances,chemiluminescence,polymerase chain reaction and gene mutational site detecting method,were applied to the diagnosis and treatment of liver disease,thus improving the degree of precision,accuracy,sensitivity,specificity and detection speed and satisfying the requirement of diagnosis and treatment maximatily.

Objective To study the effect of finasteride on hematuria associated with benign prostate hyperplasia. Methods We evaluated 61 patients with intermittent hematuria who were randomized to a finasteride treated group or control group.Routine urinoscopy was carried out once a week in all the 61 patients. Results In the untreated control group hematuria recurred in 20/31 within a year but in only 7/28 in the finasteride group,which was a statistically significant difference (P

Objective To investigate the value of combined use of misoprostol and intravenous mixture of pethidine and droperidol during operative hysteroscopy. Methods A total of 168 cases scheduled for hysteroscopy were divided into 4 groups: Group Ⅰ (42 cases) was transvaginally given 0.4 mg misoprostol as monotherapy; Group Ⅱ (41 cases) was given 0.4 mg transvaginal misoprostol in combination with intravenous mixture of 50 mg pethidine and 2.5 mg droperidol; Group Ⅲ (43 cases) was given 0.4 mg transvaginal misoprostol combined with intravenous mixture of 100 mg pethidine and 5 mg droperidol; and Group Ⅳ (42 cases), intravenous mixture of 100 mg pethidine and 5 mg droperidol. The heart rate, breath rate, blood pressure, oxygen saturation, cervical responses, VAS scores and complications were assessed respectively. Results There were statistically significant differences regarding the degree of cervical dilatation, the operation time, and the used volume of cavity-distention media between the Group Ⅳ and the Group Ⅰ, Group Ⅱ, and Group Ⅲ, respectively (P0.05). Significant differences were observed in the analgetic effect in the 4 groups (?~2=86.325,P=0.000), among which the Group Ⅱ and Group Ⅲ were superior to the other two. All patients in the 4 groups represented a stable respiratory process and blood circulation, with no significant differences in the rate of adverse effects (?~2=1.649, P=0.648). Conclusions Transvaginal application of misoprostol offers a convincing effect for cervical dilatation. Combined use of misoprostol and mixture of 50 mg pethidine and 2.5 mg droperidol gives a satisfactory analgetic effect during operative hysteroscopy.

Objective To investigate the mid-term efficacy of percutaneous laser disc decompression(PLDD) for patients with lumbar disc herniation.Methods Between May 2001 and December 2006,a total of 122 patients with simple lumbar disc herniation underwent PLDD in our hospital.Under local anesthesia,the operaiton was performed using a laser diode with the patients in supine position.A C-arm X-ray system was employed to guide the surgery.Results The patients were followed up for 6 to 60 months(mean 36.5 months).Accroding to the MacNab Criteria,60 patients achieved excellent outcomes,39 were good,15 were fair,and 8 were poor.The rate of exellent and good outcomes was 81.1%(99/122).No patient had postoperative complications.Conclusions PLDD is effective and safe for patients with simple lumbar disc herniation.The cases recover quickly after the operation,because the procedure is minimally invasive.

Objective To improve the diagnosis and differential diagnosis of adrenal incidentalomas for the optimal management of adrenal masses. Methods The clinical data of 52 cases (35 men and 17 women;mean age,42 years;age range,23-65 years) of adrenal incidentalomas was reviewed.Among them, 33 cases had tumors ≥ 3.0 cm in diameter.None of 52 cases had clinical symptoms and signs of hypertension and endocrine dysfunction.All of the tumors were incidentally found by ultrasonography or CT on regular physical examination.A series of blood chemical examinations (including electrolytes,adrenal cortical and medullary metabolic biochemistry),abdominal ultrasound and CT were performed in all 52 cases;and MRI,PET and  131 I-MIBG imaging were performed in 6,2 and 1 cases,respectively.Surgery and postoperative pathological study were performed in all. Results Postoperative pathology showed adrenocortical adenoma in 27 cases,adrenal metastatic carcinoma in 7,adrenal pheochromocytoma in 5,adrenal cyst in 4,primary aldosteronoma in 3,adrenal neurinoma in 2,adrenocortical carcinoma in 1,retroperitoneal neuroblastoma in 1,retroperitoneal primary lymphoma in 1 and adrenal ganglionic fibroma in 1.Diagnoses based on the results of imaging examinations and laboratory tests were correct in 14 cases (27%).Of the 10 patients with malignancies,5 with primary diseases died 6-10 months after chemotherapy and 5 were lost to follow-up.The remaining 42 patients were followed up for 6 months to 9 years with no evidence of recurrence. Conclusions Multidisciplinary approaches are required to identify and differentiate adrenal incidentalomas, and early surgical treatment is suggested.

Objective To analyze the early clinical efficacy of alendronate sodium on residual pain after vertebral angioplasty for osteoporotic vertebral compression fractures.Methods By drug single treatment method,a total of 30 elderly patients with residual pain after vertebral angioplasty for osteoporotic vertebral compression fractures were included.Patients were randomly divided into 2 groups:the treatment group and the control group.All patients were given calcium carbonate D3 treatment,and the treatment group was given alendronate sodium treatment additionally.6 weeks after the treatment,visual analogy score (VAS) was used for the evaluation of pain relief.Results All patients were followed up for 6 weeks.At the end of follow-up,the rates of pain relief were 86.8％ in treatment group and 13.2％ in control group.There was a significant difference in visual analogy scores between the treatment group and the control group[(0.51±0.32),(1.59±1.21) ; t=3.4,P＜0.001],which showed the degree of pain relief was higher in the treatment group than in control group.Conclusions Alendronate sodium is effective and reliable for the treatment of residual pain after vertebral angioplasty for osteoporotic vertebral compression fractures.This study provides the scientific reference for the treatment and research of residual pain after vertebral angioplasty for osteoporotic vertebral compression fractures.

Objective To investigate the effects of different BIS values on postoperative cogni-tive dysfunction (POCD)and S100βprotein(S100β)in the early stage of postoperation.Methods Fifty patients who were scheduled for selective abdominal surgery under general anesthesia (male 34 cases, female 1 6 cases,aged 65 to 75 years,ASA Ⅰ or Ⅱ)were randomly divided into two groups:light anesthesia group (group L,n =25,BIS value was maintained at 50 to 59)and deep anesthesia group (group D,n =25,BIS value was maintained at 30 to 39).BP,HR,SpO 2 ,ECG,PET CO 2 ,inhaled anes-thetic concentration and BIS values were recorded on time points of 5 minutes after the patients ente-ring the operating room (T0 ),before endotracheal (T1 ),intubation (T2 ),incision (T3 ),two hours after incision (T4 ),three hours after incision (T5 )and at the end of surgery (T6 ).The procedure du-ration,anesthesia time,dosages of propofol,fentanyl,midazolam and VAS scores on 1 d after sur-gery were also recorded.Blood samples were collected on time points of 10 min before anesthesia,im-mediately after surgery and 24,48 h after operation.S100β concentration were detected.Mini-mental State Examination (MMSE)score and Trail Making Test (TMT)completion time were recorded on 1 d before surgery and 1,3,7 d after surgery.Results BIS value of group D were lower than group L on T2 ,T3 and T4 .The propofol dosage of group D was significantly greater than that in group L (P <0.05 ).The concentration of serum S100βincreased significantly immediate and 48 h after operation in both groups compared with 10 min before anesthesia(P < 0.05).It was still higher 24 hours after op-eration than before anesthesia.But there was no statistic difference.Compared with the end of surger-y,the concentration of serum S100βin two groups on 24 h after surgery were significantly decreased (P < 0.05 ).The concentration of serum S100β in group L on the end of surgery and 24 h after surgery were higher than that in group D significantly (P <0.05).Compared with 1 d before surgery, postoperative 1 d MMSE scores in two groups and postoperative 3 d MMSE score in group L de-creased significantly (P <0.01).Compared with postoperative 3 d,postoperative 7 d MMSE score in group L increased significantly (P <0.01).Postoperative 1,3 d MMSE score in group D were signifi-cantly higher than group L (P <0.05).Compared with 1 d before surgery,TMT completion time in two groups on 1 d after surgery were significantly prolonged (P <0.01 ).Compared with 1 d after surgery,TMT completion time in two groups on 3 d after surgery were significantly shortened (P <0.01).Compared with 3 d after surgery,TMT completion time in group L on 7 d after surgery was significantly shortened (P <0.01 ).TMT completion time in group D on 1,3 d postoperative were significantly shorter than group L (P <0.05).POCD incidence of group D on 1 d after surgery was lower than that in group L (P < 0.05).Conclusion Different depth of anesthesia can ensure hemo-dynamic balance in old patients during surgery and after surgery.When BIS value was maintained at 30 to 39,it had lower S100βprotein levels,lower incidence of early POCD and a lesser degree of post-operative cognitive dysfunction.

Invasion of glioma is a complex process with multiple steps and multiple factors,including the inhibition of tumor cell adhesion,the degradation of extracellular matrix,the promotion of tumor cells migration and angiogenesis.The abnormal activation of Wnt/β-catenin signaling pathway is closely related to the invasion of glioma.The study of Wnt/β-catenin pathway affecting invasion mechanism of glioma will provide new ideas and targets for the treatment of glioma.