A mutation is predominant in weak D individuals,and DⅥⅢ mutation in partial D individuals.

AIM Valproate (VPA) is widely used to treat epilepsy. Long-term treatment of women with VPA has been reported to be associated with a PCOS-like syndrome. The aim of the present study was to investigate the effects of clinically relevant concentrations of valproate on steroidogenesis and steroidogenesis acute regulatory protein (StAR),cholesterol side-chain cleavage enzyme (P450scc)and cytochrome P450 aromatase (P450arom) mRNA expression in human luteinized granulosa cells in vitro. METHODS Human luteinized granulosa cells were isolated from oocyte retrieval of in vitro fertilization procedure and were cultured with DMEM medium and treated with various concentrations of valproate (0, 100, 250 mg?L -1), the culture media was collected after 2 days for progesterone and estradiol measurements by standard radiommunoassay, StAR, P450scc and P450arom mRNA in granulose cells were detected by fluorescent real-time RT-PCR (TaqMan assay). RESULTS Valproate caused significant increase of progesterone (P

Objective To observe the effect of Yun-Pi Prescription in different dose on small intestinal function of splenic asthenia rats, so as to explore mechanisms of Yun-pi Prescription in treatment of children apositia. Methods Experimental rats were randomly divided in to six groups:the control group, the model group, the positive control group (Xiao shi Jian er Syrup group), Yun-Pi Prescription in high, middle and low dose group. Measure the changes of body weight and food intake, observe excretory rate of D-xylose of rats. Results Yun-Pi Prescription could increase body weight and food intake, improve excretory rate of D-xylose in splenic asthenia rats. Conclusion Yun-Pi Prescription could improve the small intestinal absorptive function of splenic asthenia rats with dose-effect relationship.

Objective To compare the clinical efficacy of seretide (50 μg/500 μg salmeterol/fluticasone propionate) with seretide (50 μg/250 μg salmeterol/fluticasone propionate) in single inhaler in the treatment of patients with stable status moderate to severe chronic obstructive pulmonary disease (COPD)Methods Sixty patients with COPD were randomly divided into the treatment and the control groups.Baseline treatments were similar in all patients,patients in the treatment group received seretide (50 μg/500 μg) while the control group received seretide (50 μg/250 μg) inhalation once every 12 hours for 24 weeks Before and after the therapeutic course,tests for lung function in patients of the two groups were conducted and compared with each other.Clinical symptoms and physical signs were graded by questionnaire.Results There was no significant difference on indexes of lung function between the two groups at baseline (P ＞ 0.05).After treatment,the score of clinical symptoms and signs in the treatment group was lower than that in the control group ((4.0 ± 0.5) vs.(4.8 ± 0.3),t =2.63,P ＜ 0.05).Six minutes walking distance was longer in the treatment group than that in the control group ((451.6±22.9) meter vs.(401.2 ±25.4) meter,t =2.51,P ＜0.05).The levels of forced exhaled gas volume 1 (FEV1),FEV1/forced vital capacity (FVC) and FEV1/pred in the treatment group were higher than those in the control group ([FEV1:(2.18 ± 0.38) L vs.(1.78 ± 0.45) L;FEV1/pred:(63.19 ±9.08)％ vs.(57.19 ±9.25)％; FEV1/FVC％:(73.8 ±5.6)％ vs.(67.3 ± 11.5)％ ;P ＜ 0.05).Conclnsion High dosage of seretide had better effect in the treatment of stable moderate and severe COPD,and can obviously improve patients' lung function,clinical symptoms and quality of life.

Objective To investigate the analgesic effect of intrathecal(IT)human bone marrow mesenchymal stem cells(hMSC)genetically modified with human proenkephalin gene(PENK)in a rat model of neuropathic pain.Methods Forty male SD rats weighing 160-180 g in which IT catheters were successfully implanted without complication were randomly divided into 4 gorups(n = 10 each): group A normal control;group B neuropathic pain(NP);group C NP + hMSC-pBABE and group D NP + hMSC-PENK.Neuropathic pain was induced with chronic constrictive injury(CCI).Four loose ligatures were placed on the main stem of sciatic nerve with 4-0 chronic catgut.IT normal saline 10 μl,hMSC-pBABE cell suspension 10 μl(2 × 108-3 × 108/μl)and hMSCPENK cell suspension 10 μl(2 × 108-3 × 108/μl)were injected in group B,C and D respectively on the 3rd day after operation.Paw-withdrawal latency(PWL)to noxious thermal stimulation was measured before(baseline)and at 3,5,7,9 and 14 d after operation.The animals were killed on the 14th day after last PWL measurement.RNA was extracted from the spinal cord for determination of proenkephalin mRNA expression.Results PWL was significantly decreased after operation as compared with the baseline values before operation in group B,C and D.PWL was significantly longer at 7,9,14 d after operation in group D than in group B and C but there was no significant difference in PWL after operation between group B and C.PENK mRNA expression was significantly lower in group B and C than in group A,but was significantly higher in group D than in group B and C.There was no significant difference in PENK mRNA expression between group B and C.Conclusion Intratheccal human bone marrow mesenchymal stem cells genetically modified with human proenkephalin gene can relieve neuropathic pain in rats.

BACKGROUND: Mesenchymal stem cells (MSCs) are commonly observed under the scanning electron microscope, transmission electron microscope and inverted microscope. However, above-mentioned observation methods have disadvantages on observing ultrastructure of cell membrane and cytoskeleton. OBJECTIVE: To establish a more effective and appropriate method to isolate, culture and identification of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro and to study the ultrastructure of osteogenic differentiation with the atomic force microscope. METHODS: The hUCMSCs were isolated from human umbilical cord by digested with collagenase. After serial subcultivation in vitro, the stem cells were passaged, and osteogenic differentiation was determined by alkaline phosphatase calcium-cobalt staining and alizarin red staining. hUCMSCs immunophenotype was measured by Flow cytometry.The membrane surface ultrastructure of osteogenic differentiation was observed by Atomic Force Microscope before and after induction. RESULTS AND CONCLUSION: The isolated hUCMSCs by digested with collagenase was efficient. After seeded 24 hours, the adherent cell showed spindle shape, polygonal shape and fibroblast-cell-like shape and the size of hUCMSCs was homogeneous. Flow cytometry analysis revealed that CD29, CD44, CD105 were highly expressed on the surface of passages 3 cells, but there was negative for CD34, CD45 and HLA-DR. These cells were high positive for alkaline phosphate staining and also showed significant calcium node using alizarin red staining after 4 weeks culture induction of osteogenic differentiation. Under atomic force microscopy, undifferentiated stem cells demonstrated insignificant microtubule protrution in a parallel distribution following analysis of cytoskeleton before and after differentiation.

Objective To construct h n bone marrow mesenchymal stem cell line genetically modified with human proenkephalin gene. Methods The packaging cell line Phoenix-293T was transfected with the recombinant pBABE-PENK vector to aquire virus. The recombinant virus was then collected and used to infect hMSCs. Stable expression of proenkephalin gene and leucine enkephalin protein and the concentration of leucine enkephalin protein were detected by RT-PCR, immunofluorescence and ELISA respectively. Results The expression of proenkephalin gene and leucine enkephalin protein were significantly up-regulated in the hMSC-PENK cells, and the concentration of leucine enkephalin protein was also increased in the culture medium. Conclusion A human mesenchymal stem cell line that expresses proenkephalin gene and secrets enkephalin was successfully established.

Objective To investigate the expression and significance of MMP-9 and Ki67 for predicting the progres-sion and prognosis of pancreatic cancer. Methods S-P immunohistochemical method was used to detect the expressions of MMP-9 and Ki67 in 100 pancreatic cancer tissue specimens. The relationship between the expressions of MMP-9 and Ki67 and patient age, tumor size, lymph node metastasis, tumor differentiation, clinical stages and prognosis were analyzed. Re-sults There were higher expressions of MMP-9 protein 46%(46/100) and Ki67 protein 53%(53/100) in 100 samples of pancreatic adenocarcinoma. And the expressions of MMP-9 and Ki67 were inversely associated with tumor differentiation, clinical stages, and lymph node metastasis of pancreatic cancer (P＜0.05). There were no significant differences in the ex-pressions of MMP-9 and Ki67 between patient age and tumor size. The expressions of MMP-9 and Ki67 were positive corre-lated (rs=0.405,P＜0.05). Moreover, the overall survival rates were correlated with patient age, lymph node metastasis, tumor differentiation and the expression of MMP-9, but no correlation with tumor size, clinical stages, and the expression of Ki67. Conclusion The expressions of MMP-9 and Ki67 were associated with pancreatic cancer progression. And the detection of expression of MMP-9 may have practical value in prognosis in patients with pancreatic cancer.

Objective Dynorphins have advantages in powerful analgesic effect, high safety, no respiratory depression and no addiction, which is the emphasis of analgesic research at present. The aim of the article was to explore the expression of lentivirus-mediated rat prodynorphin gene in bone marrow mesenchymal stem cells( BM-MSCs) and contribute to the subsequent studies on bio-logical analgesia in cancer pain of rat model. Methods BM-MSCs were isolated and proliferated using the adherence screening meth-od, and further identified by flow cytometry, adipogenic and osteogenic differentiation experiments. The PDYN lentiviral vectors in rats were transfected into BM-MSCs after construction. The expression of green fluorescent protein (GFP) was detected under inversion fluo-rescence microscope and the best multiplicity of infection ( MOI ) of virus was screened by western blot. There are three groups in the ex-periment: blank group, experimental group ( PCDH-CMV-PDYN-EF1-copGFP ) and empty vector group ( PCDH-CMV-MCS-EF1-copGFP). PYDN gene was determined by qPCR and western blot, while DYN protein was detected by immunochemical method.Results BM-SMCs were in longspindle-shape and fibrocyte-like adherent growth, most in expression of CD29, CD44 and CD90, and a few in CD45. The oil red-O staining of the induced cells by adipogenic differentiation was positive. The mineralized nodules formed in the induced cells by osteogenic differentiation were orange after alizarin red staining. Flow cytometry detection showed the positive rates of CD29, CD90, CD44 and CD45 were respectively (99.80±0.19)%, (99.62±0.24)%, (96.86±1.27)%, (0.82±0.06)%, while after transfection the positive rates were (99.59±0.34)%, (98.06±1.27)%, (95.23±0.71)%, (10.23±0.59)%, representing no sig-nificant difference before and after PDYN transfection. Lentiviral vector of PCDH-CMV-PDYN-EF1-copGFP was successfully construc-ted after the identification of PCR amplification, cloning and sequencing. The titer of recombined lentiviruses was 5×106IU/mL. The best MOI of lentiviruses was 100 according to the results of GFP and western blot. Western blot and qPCR suggested PDYN gene signif-icantly increased in BM-MSCs after lentiviral transfection ( P<0.05) , and immunohistochemical staining indicated DYN protein also in-creased greatly. Conclusion BM-MSCs are successfully cultured and the overexpressed rat PDYN gene lentivirus vector is also suc-cessfully constructed;PDYN gene is highly and stably expressed and DYN protein is secreted in BM-MSCs.

Objective To evaluate the surgical treatment of splitting right hepatic duct with hepatolithiasis and stenosis. Methods The clinical data of 38 patients with splitting right hepatic duct and hepatolithiasis treated by operation were analyzed retrospectively. Results All the patients underwent operation. operative procedures were as follows: (1) in situ cholangioplasty of splitting right hepatic duct in 7 cases;(2) fenestration of splitting right hepatic with adjacent hepatic duct in 9 cases; (3) bilioplasty of splitting right hepatic duct with adjacent bile duct in 8 cases; (4) hepatic lobectomy or segmentectomy of splitting right hepatic duct in 14 cases. Postoperative complications developed in 6 cases, which were cured conservatively. There was no perioperative mortality. All patients were followed up for 5～16 years(averaged 9.2 years). Excellent rate was 78.9%,and residual stones were found in 26.3% of the patients . Conclusions Accurate localization and appropriate operation may get satisfactory result in treating patients with splitting right hepatic duct with hepatolithiasis and stenosis.