BACKGROUND: Transfection is the most important beginning component of research about gene function. It is a problem to find a transfection agent with high efficiency and safety. Nanosized materials have high surface activity, are easy to be modified and easier to pass the biomembrane. Researchers are studying how to use nanosized materials as a transfection agent.OBJECTIVE: To study the transfection efficiency of different basic polymers with different molecular mass and degree of substitute, and to find the optimized transfection agent.DESIGN: Controlled study.SETTING: Institute of Genetic Engineering, Southern Medical University.METHODS: This study was performed in the Institute of Genetic Engineering, Southern Medical University from March 2006 to June 2007. Using the lipofectamine reagent as the positive control, we transfected siRNA (0.2 nmol/L), FITC-labeling targeting bcl-2, by nine nanometers (polylactic acid-polyglycolic acid, chitosan-poly-caprolactone, polyethyleneimin-macrogol) into leukemic cell K562 cultured without serum. Six hours post-transfection, 20% FBS serum was added. The cell proliferation was measured at 24, 48 and 72 hours by using the MTT method. After transfecting for 48 hours, the cells were collected to detect transfection ratio by using fluorescent microscopy, apoptosis ratio and expression of K562 and bcl-2 protein by flow cytometer (FCM). RESULTS: ① Fluorescence microscope detection showed there were significant differences of transfection efficiency between different materials (P < 0.05). Moreover there were statistical differences between different degrees of substitute, although they were the same material (P < 0.05). ② MTT method indicated the cell proliferation ratio was positively related to transfection ratio. ③ Flow cytometry results showed the suppression of expression of targeting gene and apoptosis ratio were positive correlated with transfection ratio. CONCLUSION:The nanometer poly-ethylene glycol combined with poly-ethylene imine (PEG-PEI), whose molecular mass is 1 800/2 000 and degree of substitute is 29%, has a high efficiency and low toxicity.

Objective To investigate T cell receptor(TCR)variable β(BV)chain usage at the maternal-fetal interface and explore the relationship between the skewed TCR BV usage and unexplained recurrent spontaneous abortion(BSA).Methods Eighteen cases with unexplained RSA,together with matched 41 women with normal pregnancies in first trimester from Renji Hospital,Shanghai Jiao Tong University were studied.A high-resolution spectrum typing analysis of complementarity-determining region 3 (CDR3)was used to detect and compare the degree and frequency of TCR BV family expression in deciduas between RSA patients and normal controls.Results(1)The expression degree of BV19(0.029±0.031 vs.0.013±0.010,P=0.038)in RSA group showed a higher usage,while BV5.2(0.040±0.035 vs.0.067±0.052,P=0.046)showed a significantly lower usage when compared with normal controls.No significant difference in the expression of the other TCR BV families between RSA and controls were observed(P>0.05).(2),TCR BV2,3,6,and 7 were the four most common BV families in deciduas of patients with RSA and normal controls,whose frequencies were all mors than 50%.In RSA group,higher frequencies of BV15 (33.3%vs.7.3%,P=0.018),BV19(38.9%vs.14.6%,P=0.049)and BV20(33.3%vs.7.3%,P=0.018)were observed;meanuhile lower frequencies of BV4(33.3%vs.65.9%,P=0.026)and BV7 (66.7%vs.92.7%.P=0.018)distributions were observed.The other TCR BV families did not display significantly different freqencies of distribution(P>0.05).Conclusions It is suggested that a significant skewed TCR BV family occurs at the maternal-fetal interface in patients who undergo abortion.The specific skewed usages of TCR BV might be associated with the susceptibility to unexplained pregnancy loss.

Objective To study CT and MRI findings of the eclamptic encephalopathy in plateau.Methods CT and MRI findings in 28 patients in plateau with eclamptic encephalopathy were retrospectively analyzed.Results CT and MRI appearances of eclamptism were as follow:normal in brain in 3 cases;cerebral edema in 18 cases,including slight encephaledema in 2 cases,focal encephaledema in 11 cases and diffuse encephaledema in 5 cases;cerebral hemorrhage in 6 cases and sinovenous thrombosis in 1 case.Conclusion CT and MRI scan are of significant value in diagnosis and treatment of eclamptic encephalopathy in plateau.

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 To explore the effect of blocking BTLA-HVEM (herpesvirus entry mediator-B and T lymphocyte attenuator) pathway on dendritic cell function and the related immunological mechanisms. Methods Murine BTLA extracellular domain eukaryotic expression vector psBTLA was constructed by gene recombination and transfected CHO by Lipofection method. Mouse bone marrow cells were induced to differentiate into DCs by GM-CSF plus IL-4. Expression of BTLA and HVEM on DCs was detected after HSPT0-TC-1 peptide complex stimulation by FACS. Expression of BT-1 and secretion of IL-12 were detected after HSP70-TC-1 peptide complex plus psBTLA transfected CHO culture supernatant stimulation on DCs. Pretreated DCs co-cultured with the same genetic background mouse splenocytes and lymphocytes proliferation and cytokine secretion were detected. Effect of psBTLA gene transfer in vivo on BT-1 expression of DCs and tumor growth on tumor-bearing mice was detected. Results Extracellular domain of murine BTLA was successfully constructed, psBTLA stable transfection CHO cells were obtained and expression of BTLA extracellular domain(sBTLA) was detected the in its culture supernatant. BTLA and HVEM expression of DCs were increased after stimulation by the antigen peptide complex. When DCs were treated with antigen peptide complex plus culture supernatant containing sBTLA, B7-1 expression and IL-12 secretion were increased. Co-cultured with splenocytes, lymphocytes proliferation and cytokine secretion, such as IL-2 and IFN-γ,, were also increased. Gene transfection with psBTLA in vivo promoted B7-1 expression on DCs and inhibited cervical cancer cells growth. Conclusion Blockade of BTLA-HVEM inhibitory pathway with sBTLA can further improve DCs function, activation of lymphocytes and promote antitumor immune response.

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.

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 explore application of nursing intervention of children's fun mode m anesthesia induction of preschool children.Methods A total of 180 preschool children undergoing elective surgery in our hospital were randomly divided into control group and observation group according to the random number table method,with 90 cases in each group,the control group was given routine nursing,and the observation group with children's fun mode nursing intervention,the curative effect is compared between the two groups.Results Before operation,there was no significant difference between the two groups in the improvement of the perioperative anxiety scale (mYPAS) score in the groups (P ＞ 0.05).During induction of anesthesia,mYPAS score of the two groups before operation were significantly higher than one day before surgery,and the differences were statistically significant (P ＜ 0.05),and anxiety score in the observation group was significantly lower than the control group (P ＜ 0.05).Before one day of operation,there was no significant difference in systolic blood pressure (SBP) and diastolic blood pressure (DBP) between the two groups (P ＞0.05).During induction of anesthesia,the SBP,DBP of two groups were significantly higher than one day before operation,and the differences were statistically significant (P ＜ 0.05),and the observation group had lower SBP and DBP than the control group,the difference was statistically significant (P ＜ 0.05).Before one day of surgery,there was no significant difference in heart rate (HR) between two groups (P ＞ 0.0 5).During induction of anesthesia,the two groups had higher HR than one day before operation (P ＜ 0.05),and the observation group had lower HR than the control group (P ＜ 0.05).The observation group during anesthesia induction had lower cooperation scale (ICC) score than the control group(P ＜ 0.05).Conclusion Nursing intervention of children's fun mode for preschool children can improve children's blood pressure,and has less influence on HR so as to improve cooperation of anesthesia induction.

Objective To explore application of nursing intervention of children's fun mode m anesthesia induction of preschool children.Methods A total of 180 preschool children undergoing elective surgery in our hospital were randomly divided into control group and observation group according to the random number table method,with 90 cases in each group,the control group was given routine nursing,and the observation group with children's fun mode nursing intervention,the curative effect is compared between the two groups.Results Before operation,there was no significant difference between the two groups in the improvement of the perioperative anxiety scale (mYPAS) score in the groups (P ＞ 0.05).During induction of anesthesia,mYPAS score of the two groups before operation were significantly higher than one day before surgery,and the differences were statistically significant (P ＜ 0.05),and anxiety score in the observation group was significantly lower than the control group (P ＜ 0.05).Before one day of operation,there was no significant difference in systolic blood pressure (SBP) and diastolic blood pressure (DBP) between the two groups (P ＞0.05).During induction of anesthesia,the SBP,DBP of two groups were significantly higher than one day before operation,and the differences were statistically significant (P ＜ 0.05),and the observation group had lower SBP and DBP than the control group,the difference was statistically significant (P ＜ 0.05).Before one day of surgery,there was no significant difference in heart rate (HR) between two groups (P ＞ 0.0 5).During induction of anesthesia,the two groups had higher HR than one day before operation (P ＜ 0.05),and the observation group had lower HR than the control group (P ＜ 0.05).The observation group during anesthesia induction had lower cooperation scale (ICC) score than the control group(P ＜ 0.05).Conclusion Nursing intervention of children's fun mode for preschool children can improve children's blood pressure,and has less influence on HR so as to improve cooperation of anesthesia induction.

Objective To improve the utilization of operation rooms and to reduce the preoperative preparation time of elective surgery under general anesthesia as well as working efficiency. Methods Launched the QCC activity with the theme of"shortening the preoperative preparation time of elective surgery under general anesthesia" and investigated 4673 surgery cases from May to November 2013. Analyzed the reasons, found out measures and took them into application. Results The mean time of preoperative preparation of elective surgery under general anesthesia reduced from 60. 29 minutes to 39. 10 minutes (P <0. 05). Conclusions The application of QCC can reduce the preoperative preparation time of elective surgery under general anesthesia and improve the work efficiency, intelligence of members and their teamwork spirit.