Objective: To establish clear cell renal cell carcinoma (ccRCC) cell lines from clinical ccRCC specimens of Han nationality in China and to characterize the biological features. Methods: From 2005 to 2007, fresh surgical samples of ccRCC were obtained from 43 patients; the samples included primary tumor in situ, osseous metastasis, lymph node metastasis, and cancerous embolus. The samples were cultured in vitro using explant-culture method within 30-60 min after surgery. Analysis on cell growth and colony-forming efficiency was recorded for the lines which were passaged for over 50 generations. Chromosome examination, pathological examination and tumorigenesis in NOD-SCID mice were used to determine their malignancy. Flow cytometry was used to determine expression of CA9 and CD133. Results: Most of the primary cells could only be passaged for less than 5 generations; 5 lines could be serially passaged for over 5 passages, 3 lines for over 10 passages, and only 2 lines could be stably passaged. One line,named RCC05-TXJ, was from osseous metastatic ccRCC and had been serially passaged for 110 generations in 21 months; the average doubling time was 19.2 h,average chromosome number was 75,and colony forming efficiency was 41%. Another line,named RCC05-ZYJ,was from primary ccRCC specimen and had been serially passaged for 160 generations in 18 months; the average doubling time was 16.5 h,average chromosome number was 55,and the colony forming efficiency was 37%. Immunohistological analysis demonstrated that both lines expressed CA9 and CD133. Flow cytometry analysis found that expression levels of CA9 and CD133 increased with the passages. Both RCC05-ZYJ and RCC05-TXJ lines were able to form tumor and to metastasize in NOD-SCID mice; however, their metastatic ability was obviously different. Conclusion: We have established 2 ccRCC cell lines with different metastatic potentials from the clinical ccRCC specimens of Han nationality in China. The ratio of tumor stem cells increases with the passages.

Objective: To observe the expression of CD133 in the clear cell renal cell carcinoma (RCC) and to investigate the related drug resistance. Methods: Flow cytometry and immunohistochemical method were used to examine the expression of CD133 in metastatic RCC cell line RCC05-TXJ, low metastatic RCC cell line RCC05-ZYJ and two clinical non-metastatic RCC primary cultures isolated from a male and a female patient. The four cell lines were treated with IFN-α and 5-FU and the viability of cells were examined by MTT assay before and after treatment. Results: Flow cytometry showed that RCC05-TXJ and RCC05-ZYJ expressed CD133. The in situ carcinomas of male and female patients hardly expressed CD133. Immunohistochemistry examination showed that the local membrane of RCC05-TXJ and RCC05-ZYJ cells expressed CD133. RCC cells of female and male patients hardly expressed CD133. RCC05-TXJ and RCC05-ZYJ cells exposed to IFN-α or 5-FU showed a rebound of survival 24 h after withdrawal of drugs. The survival rates of RCC cells of male and female patients kept at a low level after withdrawal of drugs. Conclusion: There are a small number of CD133+ RCC cells in RCC patients, with a property similar to tumor stem cells, which might be one of the important factors influencing the immunotherapy and chemotherapy of tumors.

Objective To investigate the role of extracellular signal-regulated kinase(ERK)signal pathway in the spinal cord in the development of chronic morphine tolerance.Methods Thirty healthy male SD rats weighing 300-350 g in which intrathecal(IT)catheters were successfully implanted without complication were randomly divided into 3 groups(n = 10 each): group Ⅰ control(group C);group Ⅱ morphine tolerance(group M)and group Ⅲ morphine tolerance + PD98059(ERK upstream kinase MEK inhibitor)(group P).Morphine tolerance was induced with IT morphine 10 μg twice a day for 7 consecutive days.In group P PD98059 10 μg was injected IT at 30 min before each morphine administration.Tail flick latency(TFL)(the time between the onset of heat stimulus and voluntary tail withdrawal)was measured once a day at 30 min after first IT morphine administration and at 1 day after last IT morphine.Maximum analgesic effect(MPE)was calculated.MPE =(TFL after IT morphine - baseline TFL)/(12 - baseline TFL)× 100%.The animals were sacrificed after last TFL measurement.The L3-5 segment of the spinal cord was isolated for determination of the expression of μ-receptor and phosphorylated ERK 1/2(p-ERK1/2)by Western blot analysis and fluoroimmunoassay.Results Morphine tolerance was induced in group M and M + P.MPE was higher in group P than in group M.The expression of μ-receptor in spinal dorsal horn was significantly lower while the p-ERK1/2 expression was higher in group M than in group C.IT PD98059 significantly up-regulated μ-receptor expression and down-regulated p-ERK expression in group P as compared with group M.Conclusion ERK signal pathway is involved in the development of chronic morphine tolerance in rats.

Objective To establish a HPLC method to determine the related substances of vitamin B6 for injection.Methods HPLC method was adopted with DiamonsilTM C18,5μm 250mm×4.6mm;the mobile phrase was length was 291nm.Results The calibration curve was linear in the concentration range of 600～1400μg/ml(r=0.9998).Conclusion The method is simple,rapid,accurate and reliable.As the supplement method of active pharmacopoeia,it can effectively control the quality of the product.

Objective To investigate the role of glucocorticoid receptor (GR) in the induction of neuronal apoptosis in spinal dorsal horn in chronic morphine tolerant rats. Methods Twenty healthy male SD rats weighing 300-350 g in which intrathecal (IT) catheter was successfully implanted without complication were randomized into 4 groups ( n = 5 each): group Ⅰ control ( group C);group Ⅱ morphine ( group M );group Ⅲ morphine +RU38486 (GR antagonist) (group MR) and group Ⅳ morphine + dexamethasone (GR agonist) (group MD).Normal saline 10 μl, morphine 10 μg, morphine 10 μg + RU38486 2 μg and morphine 10μg + dexamethasone 4 μg were injected IT twice a day at 8:00 and 20:00 for6 consecutive days in group C, M, MR and MD respectively. Tail flick latency (TFL) to a thermal nociceptive stimulus was measured every day at 30 min after IT administration in the morning (8:00). Hyperalgesia was considered to be a sign of morphine tolerance. The animals were killed at 7 days after IT drug administration. The L3-5 segment of the spinal cord was isolated for determination of neuronal apoptosis in spinal dorsal horn by TUNEL staining. Apoptotic index was calculated ( the number of apoptotic neurons/the total number of neurons × 100% ). Results TFL was significantly prolonged at day 1 and 3 of IT morphine 10 μg twice a day and returned to baseline at day 5 and 7 indicating morphine tolerance. RU38486 inhibited while dexamethasone enhanced morphine tolerance. IT morphine significantly increased the number of apoptotic neurons in spinal dorsal horn. Morphine-induced neuronal apoptosis was decreased by IT RU38486 and increased by IT dexamethasone. Conclusion Glucocorticoid receptors may be involved in morphine tolerance by inducing neuronal apoptosis in spinal dorsal horn.

Objective To investigate the clinical and endoscopic features of ischemic colitis (IC),to provide a reference for clinical diagnosis and treatment.Methods A retrospective analysis of 62 inpatients who were diag-nosed as IC was performed.The patients'clinical data,clinical manifestations,complications,laboratory tests,imaging tests,colonoscopy,pathology,diagnosis,treatment,follow -up and other information were analyzed.Results Of 62 patients,there were 1 7 males and 45 females,mean age was (66.1 ±1 0.7)years old,more than 60 years of age there were 47 cases (75.81 %),of which 38 cases (61 .29%)showed the presence of one or more underlying disea-ses.All patients had acute onset,57 cases (91 .94%)had abdominal pain,bloody stools occurred in 56 cases (90.32%).Colonoscopy revealed 60 cases as a transient type,2 cases of stenosis,no gangrene type,lesions were mainly left colon.All patients were given fasting,anti -infection,microcirculation improvement and intravenous nutri-tional support,with no serious complications or transfer surgery occurred,colonoscopy was reviewed 4 -8weeks, 62 cases were healing or had improvement,2 patients in the 3 -year observation period was readmitted attack,the recurrence rate was 3.23%.Conclusion Ischemic colitis is more common in older people,to strengthen the aware-ness and vigilance of the disease,early diagnosis based on colonoscopy as a primary diagnostic modality and prompt treatment has positive significance.

Clinical medicine is a practical discipline.Students combine theoretical knowledge with practical operation and develop clinical thinking at stage of clinical practice.From the perspective of education administration,a series of measures were carried out throughout the whole teaching in clinical internship in our university.Students were trained to gain the abilities of combing knowledge with practice,analyzing and solving issues individually and thinking logically and scientifically.Through years of research and practice,remarkable achievements were gained and both clinical skill and comprehensive quality of our students were greatly enhanced.

The diameter of the mitral orifice was measured in 122 cases of rheumatic mitral stenosis when commissurotomy was performed. In 99 cases(81.25%) with a history shorter than 10 years, the diameters ranged from 0.3 to 2 cm; it was larger than 1.1 cm. in 18 cases and smaller than 0.8 cm. in 68 cases.After analyzing the interrelationship between the diameter of the mitral orifice and the clinical manifestations, ECG, the postoperative complications, and the pathological findings, it was found that the size of the mitral orifice was not always in direct proportion with either the duration of the illness or the severity of cardiac enlargement. In those patients with a mitral orifice smaller than 0.8 cm., the following criteria were often present:1. Cardiac function above Ⅲ degree.2. Repeated attacks of hemoptysis.3. Presystolic contraction on ECG.4. Postoperative complication of auricular fibrillation.5. Thrombosis on the cardiac walls detected pathologically.

Objective To evaluate the value of homocysteine(Hcy) detection by different assays in the diagnosis of hypertension complicating cerebral infarction disease .Methods Serum concentration of Hcy in the patients with single hypertension ,hyperten‐sion complicating cerebral infarction and healthy controls were detected by the fluorescence quantification assay and the enzymatic cycling assay respectively ,the application value of the two assays in the diagnosis of hypertension complicating cerebral infarction disease was evaluated by the receiver operating characteristic (ROC) curve .Results Both the two assays showed that the serum Hcy level and incidence of high Hcy in the single hypertension group and the hypertension complicating cerebral infarction group were obviously higher than those in the healthy control group (P<0 .05) ,but there were no statistically significant differences be‐tween the single hypertension group and the hypertension complicating cerebral infarction group (P>0 .05) .The ROC curve analy‐sis showed that the sensitivity and the specificity in the enzymatic cycling assay were higher ,which were 67 .5% and 85 .3% respec‐tively ,but the fluorescence quantification assay had lower sensitivity (40 .0% ) and higher specificity (97 .1% ) .Compared to the sin‐gle index test ,the sensitivity of Hcy and triglyceride combination detection had higher sensitivity (72 .5% ) and higher specificity (94 .1% ) ,which were higher than the diagnostic performance of the single index .Conclusion The increase of serum Hcy level is closely related to hypertension ,but is not a direct causal relationship with hypertension complicating cerebral infarction .The enzy‐matic cycling assay is better than the fluorescence quantitative assay in the diagnostic performance of hypertension complicating cer‐ebral infarction .The combination detection of Hcy and triglyceride conduces to improve the diagnostic sensitivity and specificity .

Objective To compare the safety between laparoscopic cholecystectomy (LC) and open cholecystectomy (OC) for benign gallbladder diseases in elderly patients. Methods A total of 120 patients above the age of 60 with indications for cholecystectomy and tolerance to general anesthesia were divided into two groups (LC Group and OC Group), in admission order of odd or even, with 60 patients in each group. Peri-operative parameters including operation time, analgetic consumption, postoperative gastrointestinal recovery time, length of duration with fluid infusion, length of bed rest, postoperative complications, and hospital stay were compared between the two groups. Serum total triiodothyronine (TT3) and thyroid stimulating hormone (TSH) levels, as well as the functional recovery of the abdominal muscles, were also recorded and compared. Results Peri-operative parameters were better in the LC Group than in the OC Group. Serum TT3 levels decreased significantly after surgery in both the LC Group (F=8.26,P=0.000) and the OC Group (F=124.70,P=0.000), with a more significant difference in the OC Group.Postoperatively,serum TSH levels decreased insignificantly in the LC Group (F=1.87,P=0.157) and significantly in the OC Group (F=27.24,P=0.000). Lasegue’s test at 7 days after surgery showed significantly higher leg-raising times in the LC Group than in the OC Group (t=3.640,P=0.000). Conclusions Laparoscopic cholecystectomy is superior to open cholecystectomy in the treatment of benign gallbladder diseases in elderly patients.