Objective The gene encoding part of HTLV(Human Tcell leukemia viruses) envelope protein gp21 was cloned and expressed in order to prepare diagnostic agent for detecting HTLV.Methods The gene encoding part of HTLV envelope protein gp21 was obtained by combining four oligonucleotides into a long DNA fragment,then cloned and subcloned into Tvector and pGEX2T respectively.Expression was performed under induction in a fusion way that target protein gene was inserted downstream the carrying protein gene of pGEX2T which makes purification of target protein convenient.At last,Target protein was purified by affinity chromatography in glutathione sepharose 4B column and its antigenicity was confirmed by immunoblotting.Results and conclusion Sequence analysis showed it was feasible to obtain a target gene by ligating short oligonucleotides;As expected,target protein was expressed which exhibited strong antigenicity after being subcloned into fusion expression vector pGEX2T.

Purpose To test the virus inactivation effect of water bath method at 60 ℃ for 10 hours and alcohol treatment for 3 hours which was used in the production of urinary trypsin inhibitor(UTI).Methods Sindbis virus,Pseudorabies virus(PRV) and poliovirus1(PV1) were used as indicated viruses in this test.After being added separately into the UTI raw material in 10% proportion,the viruses were treated with water bath at 60 ℃ for 10 hours and alcohol for 3 hours and then the samples of UTI were taken to inoculate the cell line for assay of cytopathic effect.Results The water bath at 60 ℃ for 10 hours could inactive Sindbis,PRV and PV1 in more than(6.503±0.102)LgTCID_(50),(6.42±0.158) LgTCID_(50) and(6.587±0.061)LgTCID_(50) respectively,and alcohol treatment for 3 hours could inactive Sindbis,PRV and PV1 in more than(5.88±0.204)LgTCID_(50),(6.378±0.268)LgTCID_(50) and(5.963±0.118) LgTCID_(50) respectively.No cytopathic effect was found in the cell line which was inoculated with treated samples after blind passage for three generations.Conclusion The water bath method at 60 ℃ for 10 hours and alcohol treatment for 3 hours which were used in the production of UTI had good effects on virus inactivation and the inactivation efficiency on Sindbis,PRV and PV1 was more than 6 LgTCID_(50)/mL.

OBJECTIVE: The aim of this study was to build a model to predict the risk of lymphovascular space invasion (LVSI) in women with endometrial cancer (EC). METHODS: From December 2010 to June 2013, 211 patients with EC undergoing surgery at Shanghai First Maternity and Infant Hospital were enrolled in this retrospective study. Those patients were divided into a positive LVSI group and a negative LVSI group. The clinical and pathological characteristics were compared between the two groups; logistic regression was used to explore risk factors associated with LVSI occurrence. The threshold values of significant factors were calculated to build a risk model and predict LVSI. RESULTS: There were 190 patients who were negative for LVSI and 21 patients were positive for LVSI out of 211 patients with EC. It was found that tumor grade, depth of myometrial invasion, number of pelvic lymph nodes, and International Federation of Gynecology and Obstetrics (FIGO) stage (p<0.05) were associated with LVSI occurrence. However, cervical involvement and age (p>0.05) were not associated with LVSI. Receiver operating characteristic (ROC) curves revealed that the threshold values of the following factors were correlated with positive LVSI: 28.1 U/mL of CA19-9, 21.2 U/mL of CA125, 2.58 mg/dL of fibrinogen (Fn), 1.84 U/mL of carcinoembryonic antigen (CEA) and (6.35×10⁹)/L of white blood cell (WBC). Logistic regression analysis indicated that CA125 ≥21.2 (p=0.032) and Fn ≥2.58 mg/dL (p=0.014) were significantly associated with LVSI. CONCLUSION: Positive LVSI could be predicted by CA125 ≥21.2 U/mL and Fn ≥2.58 mg/dL in women with EC. It could help gynecologists better adapt surgical staging and adjuvant therapies.
CA-125 Antigen ; Carcinoembryonic Antigen ; Endometrial Neoplasms* ; Female ; Fibrinogen* ; Gynecology ; Humans ; Infant ; Leukocytes ; Logistic Models ; Lymph Nodes ; Obstetrics ; Retrospective Studies ; Risk Factors ; ROC Curve

Objective To explore humam cytomegalovirus(HCMV) encoded microRNAs during latent infection in order to help study HCMV virology and latent infection mechanisms.Methods A model of HCMV latent infection via THP-1 cells infected with HCMV was constructed.Deep-sequencing was performed using high-resolution Solexa sequencing platform.The secondary structure of the newly sequenced miRNA was predicted by RNAFold bioinformatics software. Results HCMV encoded various miRNAs during latent infection, including miR-US25-2-3p, miR-US25-2-5p, miR-UL112, miR-US25-1, miR-UL22A and PC-5p-148467 with a predicted length of 25 bp, named hcmv-miR-US33as-5p.Conclusion HCMV can express many types of miRNAs during latent infection.

Objective To study the effect of HCV receptors′sequence on virus entry based on the two-dimensional structure and via tandem expression of HCV receptors on mouse hepatocytes.Methods The construced recombinant expression vectors pCDH-hLDLR-hSR-BⅠ-hCD81-GFP, pCDH-hLDLR-hCD81-hSR-BⅠ and pCDH-hCLDN-1-hOCLN-DsRed were cotransfected into 293FT cells with package vectors.The collected recombinant lentivirus expressing hCLDN-1-hOCLN was concentrated and attacked mouse hepatocytes.The transgenic mouse hepatocytes with tandem overexpression of CLDN-1 and OCLN were established after G418-selection.The transduced cells LSCCO/Hepa1-6 and LCSCO/Hepa1-6 were sorted via flow cytometry and puro-G418-selection after recombinant lentivirus expressing hLDLR-hSR-BⅠ-hCD81 and hLDLR-hCD81-hSR-BⅠattacked Hepa1-6 respectively.The infectivity of transduced mouse hepatocytes LSCCO/Hepa1-6 and LCSCO/Hepa1-6 to HCV was analyzed via direct-infection of serum-derived virus.Furthermore, the effect of HCV receptors′sequence on virus entry was studied.Results Both LSCCO/Hepa1-6 and LCSCO/Hepa1-6 enhanced HCV-cell binding.The transduced mouse hepatocytes LSCCO/Hepa1-6 had more HCV endocytosis.Conclusion SR-BⅠhas priority over CD81 in HCV entry in the early stage.

Subchondral insufficiency fracture (SIF) of the femoral head is one of the predominant etiologies of rapidly progressive osteoarthritis of the hip (RPOH). SIF is a rare disease that causes acute pain in the hip joint. It is most frequently found in elderly women with osteoporosis. It is often underdiagnosed or misdiagnosed as osteonecrosis of the femoral head. SIF is currently a well-established cause of RPOH; however, the deeper etiology of SIF is not clear. Good clinical outcomes have been reported for hip preservation therapy and hip replacement. SIF is not obvious radiologically in the early stage, and a T1-weighted magnetic resonance imaging shows a discontinuous low-intensity band under the articular cartilage convex to the articular surface as its characteristic manifestation. Some patients will lose the opportunity to preserve the hip joint due to symptoms such as progressive joint space narrowing and subchondral collapse within a very short period. Patients with progressive hip space narrowing and subchondral collapse on Xray should be converted to total hip arthroplasty. Based on the characteristics of the disease, surgeons need to master the clinical and radiological characteristics of SIF and strive for early diagnosis and treatment.

Objective To study the relationship between serum uric acid (UA) level and metabolic syndrome (MS) in elderly essential hypertensive (EH) patients.Methods Two hundred and one elderly EH patients were divided into hyperuricemia group (n=47) and normal UA group (n=154).Hyperuricemia was defined in males with their serum UA level ＞ 420 μmol/L and in females with their serum UA level ＞ 360 μmol/L.Relationship between serum UA level and MS in elderly EH patients was analyzed by Pearson correlation analysis and logistic regression analysis respectively.Results The age was significantly older,the waist circumference was significantly longer,the BMI and serum SCr,FPG,TG level and the incidence of MS,central obesity,high blood glucose and high TG were significantly higher while the serum HDL-C level was significantly lower in hyperuricemia group than in normal UA group.Pearson correlation analysis showed that waist circumference,BMI,serum FPG and TG level were positively related with serum UA level while serum HDL-C level was negatively related with serum UA level (P＜0.05,P＜0.01).Logistic regression analysis showed that waist circumference and serum TG level were the independent risk factors for elevated serum UA level in elderly EH patients (OR=1.080,95％ CI:1.035-1.127,P=0.000;OR=1.472,95％CI:1.021-2.122,P=0.038).Conclusion Serum UA level is closely related with MS and its components while waist circumference and serum TG level are the independent risk factors for hyperuricemia in elderly EH patients.

ObjectiveTo investigate the protective effect of modified Huangqi Guizhi Wuwutang （MHGW） on endoplasmic reticulum stress in the sciatic nerve of diabetes rats based on the pathways of inositol-requiring enzyme 1α （IRE1α） and CCAAT/enhancer-binding protein homologous protein （CHOP）. MethodSixty rats were fed on a high-sugar and high-fat diet for six weeks， followed by intraperitoneal injection of streptozotocin at a dose of 35 mg·kg^-1. Random blood glucose levels were measured three days later and rats with a sustained blood glucose level ≥ 16.7 mmol·L^-1 were included in study （n=48）. The rats were randomly divided into a model group， an α-lipoic acid group （0.026 8 g·kg^-1·d^-1）， a high-dose MHGW group （2.5 g·kg^-1·d^-1）， and a low-dose MHGW group （1.25 g·kg^-1·d^-1）. Another 10 rats were assigned to the normal group. The intervention lasted for 16 weeks. After 16 weeks， the sciatic nerve structure of the rats in each group was observed under light microscopy using Luxol fast blue （LFB） staining. Transmission electron microscopy was used to observe the ultrastructure of the sciatic nerve. Chemiluminescence method was employed to measure the serum reactive oxygen species （ROS） levels. Western blot and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to evaluate the expression of p-IRE1α protein， IRE1α mRNA， CHOP protein， and CHOP mRNA in the sciatic nerve of the rats. ResultCompared with the normal group， the model group showed elevated serum ROS levels （P<0.01）. In contrast， the serum ROS levels were significantly reduced in the treatment groups compared with those in the model group （P<0.01）. The sciatic nerve of the model group showed pathological changes compared with that in the normal group， while the treatment groups exhibited improvement in sciatic nerve pathology compared with the model group. The protein expression of p-IRE1α and CHOP in the sciatic nerve significantly increased in the model group as compared with that in the normal group （P<0.01）. However， the treatment groups showed a significant decrease in the protein expression of p-IRE1α and CHOP in the sciatic nerve compared with the model group （P<0.05， P<0.01）. Furthermore， compared with the normal group， the model group showed upregulated mRNA expression of IRE1α and CHOP in the sciatic nerve （P<0.01）， while the treatment groups exhibited a significant decrease in the mRNA expression of IRE1α and CHOP compared with the model group （P<0.01）. ConclusionMHGW can alleviate endoplasmic reticulum stress-induced cell apoptosis and improve the structure and function of the sciatic nerve in diabetes rats by inhibiting the expression of IRE1α/CHOP pathway-related proteins and mRNA， thereby preventing and treating peripheral neuropathy in diabetes.

ObjectiveTo investigate the effect of modified Huangqi Guizhi Wuwutang （MHGW） on the protein and mRNA expression of B-cell lymphoma-2-associated X protein （Bax） and cysteinyl aspartate specific proteinase-12 （Caspase-12） related to the apoptosis of sciatic nerve cells in diabetes rats to explore the mechanism of MHGW in the treatment of peripheral neuropathy in diabetes. MethodAnimal experiments were conducted. A diabetes model was induced in sixty male sprague-dawley （SD） rats by feeding on a high-sugar and high-fat diet combined with streptozotocin （STZ） intraperitoneal injection. Rats with random blood glucose levels ≥ 16.7 mmol·L^-1 for three consecutive days were considered to have successfully developed diabetes. Forty-eight rats that successfully developed diabetes were randomly divided into a model group， an α-lipoic acid group （0.026 8 g·kg^-1·d^-1）， a high-dose MHGW group （2.5 g·kg^-1·d^-1）， and a low-dose MHGW group （1.25 g·kg^-1·d^-1）， with 12 rats in each group. Another 10 rats were assigned to the normal group. Body weight and random blood glucose levels of the rats were monitored. At the end of a 16-week intervention period， the sciatic nerve conduction velocity of the rats was measured using the Key point electromyography collection system. The protein and mRNA expression of Bax and Caspase-12 in the sciatic nerve cells was detected by Western blot analysis and real-time quantitative polymerase chain reaction （Real-time PCR）， respectively. ResultCompared with the normal group， the model group showed a significant decrease in body weight （P<0.01） and a significant increase in random blood glucose levels （P<0.01）. After a 16-week intervention， compared with the model group， the high-dose MHGW group exhibited a significant increase in body weight （P<0.05）， while there were no statistically significant differences in body weight changes among the other treatment groups. Random blood glucose levels significantly decreased in all treatment groups （P<0.01）. After 16 weeks of intervention， compared with the normal group， the model group had significantly reduced motor and sensory nerve conduction velocities （P<0.01）. Compared with the model group， all treatment groups showed significant increases in motor and sensory nerve conduction velocities （P<0.05， P<0.01）. The expression of Bax and Caspase-12 proteins in the sciatic nerve cells was significantly elevated in the model group compared with that in the normal group （P<0.01）. In contrast， all treatment groups showed significant reductions in the expression of Bax and Caspase-12 proteins in the sciatic nerve cells as compared with that in the model group （P<0.01）. The expression of Bax and Caspase-12 mRNA in the sciatic nerve cells significantly increased in the model group compared with that in the normal group （P<0.01）. Compared with the model group， the α-lipoic acid group and the high-dose MHGW group showed significant reductions in the expression of Bax mRNA in the sciatic nerve cells （P<0.05， P<0.01）， while the low-dose MHGW group showed a decreasing trend in the expression of Bax mRNA. The expression of Caspase-12 mRNA in the sciatic nerve cells significantly decreased in all treatment groups （P<0.01）. ConclusionMHGW may improve and repair sciatic nerve damage in diabetes rats by inhibiting sciatic nerve cell apoptosis.