In order to adapt to the changes of medicine mode and market needs, the Nursing Department of Fujian TCM gave an elective course of nursing etiquette, and made teaching designs for the course, including the designs of teachers training, teaching objects, teaching contents and teaching methods. At the end of the course, the authors conducted questionaire, interview and examination among the students, and analyzed the data by statistical methods, and believed that the result was good. Besides, the article discussed the meaning of setting the Nursing etiquette course, and gave some suggestions on the course building.

AIM:To establish the method of determining sodium new houttuyfonate in plasma and study its pharmacokinetics in beagle dogs.METHODS:The HPLC was employed with chromatographic conditions,such as octa-silicane bond silica gel column(250 mm?4.6 mm,5 ?m)as analytical column,methanol-water-ammonium hydroxide with 4 butyls(70 ∶30 ∶0.3)as mobile phase and at the flow-rate of 1.0 mL/min.The UV detection was set at 286 nm.The pharmacokinetic parameters were calculated by DAS(ver 1.0)program and compared by statistic analysis.RESULTS:The calibration curve was linear in blood plasma over the concentration range of 0.128～25.575 mg/L(r= 0.999 2),good precision and high recoveries.The parameters were T_ max= 5.0 min,C_ max= 4.982 mg/L,T_ 1/2=8.67 min.CONCLUSION:The analytical method established in this study is sensitive and exact,and can be used to measure the pharmacokinetics of sodium new houttuyfonate in beagle dog.The sodium new houttuyfonate can metabolize quickly following intravenous injection.

Objective To investigate the effect of different promoters on the expression level of transgene containing MAR expression vector in recombinant CHO cells.Methods The CMV promoter and 3-globin MAR were amplified by PCR,then CMV promoter was replaced the SV40 promoter in pCAT1 for constructing the expression vector droved by CMV promoter.The control vectors of pCAT1 and pCAT2 without containing MAR were simultaneously transfected into the CHO cells.Then the stably transfected cell line was screened by G418.The CAT gene expression level was analyzed by ELISA.Results The expression level of CAT enzyme in the cells transfected with MAR-containing vectors was increased compared with the cells transfected by pCATG and pCAT3 vectors without containing MAR,which were increased by 1.75 and 1.25 times respectively(P＜0.05);but CAT enzyme expression level in the pCAT1 transfected cells droved by SV40 promotor with the MAR-containing expression vectors was 1.4 times higher than that in the pCAT2 vector droved by the CMV promoter(P＜0.05).Conclusion MAR can enhance the transgene expression level in stably recombinant CHO cells,and the promoting efficiency of SV40 promoter and MAR combination is superior to that of CMV promoter and MAR combination.

Liver cirrhosis is a common chronic progressive liver disease,and at present the most effective treatment for advanced liver cirrhosis is liver transplantation.However,main reasons of limiting the wide application of liver cirrhosis are liver source deficiency,expensive cost,graft rejection reaction,the complications caused by long-term application of immunosuppressant and so on.Stem cell transplantation has become a new method for the treatment of liver diseases due to its beneficial to the damaged liver tissue repair,and it can compensate part of liver function.The basis and clinical research progress,the existing problems and prospects of the bone mesenchymal stem cell transplantation for the treatment of liver cirrhosis are summarized,aiming to provide theoretical basis for the further research.

BACKGROUND: Matrix attachment region (MAR), a DNA sequence, is still bound to the nuclear matrices after chromatindigested with restriction endonuclease, not only affects expression of endogenous gene, but also overcames transgenic silence andimproves transcription and expression of exogenous gene. OBJECTIVE: To investigate the influence of ?-interferon MAR of CHO cells on the transgenic expression of chloramphenicolacetyltransferase (CAT). DESIGN, TIME AND SETTING: The opening experiment was performed at the Department of Biochemistry and MolecularBiology, Molecular Institute, Xinxiang Medical College from October 2006 to April 2007. MATERIALS: CHO cell lines were obtained from China Center for Type Culture Collection. The pCATG vector of CAT and G418screening markers were constructed by this laboratory. METHODS: Human ?-interferon MAR by PCR was digested with SacI/KpnI and BamHI/SalI, and was inserted into pCATGvector, which was propagated in Escherichia coli JM109, then extracted and purified followed by enzyme digestion analysis. Vectorof CAT expression cassette and human ?-interferon MAR by the two sides was successfully constructed, and christened aspCAT-MAR. Two methods were compared between CHO cells of pCATG transformation and CHO cells of pCATG-MARtransformation. After G418 selecting, genome DNA of cell lines of G418 was extracted, then primers for PCR to amplify the CATtarget gene fragment was designed. MAIN OUTCOME MEASURES: The activity of CAT was analyzed by ELISA method. It was also tested to see if thepCATG-MAR was stably integrated into genomic DNA in the transfected cells. RESULTS: CHO cells of pCATG transformation was screened to have 16 strains of positive cell, and CHO cells of pCATG-MARtransformation was screened to have 17 strains of positive cell. Human ?-interferon MAR could increase the CAT gene expressionby 2.8 fold. The coefficient of variation of CHO cells of pCATG transformation was 2.065 0, and coefficient of variation of CHOcells of pCATG-MAR transformation was 0.813 1. Genome DNA of stable transformation cell lines was amplified by a fragment of437 bp. The results confirmed the pCAT-MAR vector was stably integrated into genomic DNA. CONCLUSION: Human ?-interferon MAR can increase transgenic expression in CHO cells and decrease the transgenicexpression variation in different transfected cells.

Objective To observe the clinical efficacy of Wenshen Xiaochuan Paste Recipe (WXPR) in preventing bronchial asthma, and to explore the mechanism. Methods Eighty patients were randomly divided into treatment group and control group. Each group had 40 patients. The control group was treated by conventional western medicine such as inhalation of steroid, and the treatment group was given oral use of WXPR based on the treatment for the control group. Treatment for both groups covered 60 days, and the follow-up lasted 10 months. The clinical outcomes included the clinical efficacy and immune indexes of peripheral blood T lymphocyte subsets CD3+, CD4+, CD8+and CD4+/CD8+ratio, and the serum IgE level. Results (1) After treatment and 12 months later, the scores of asthma control test (ACT ) in the two groups were increased obviously as compared with those before treatment, and there were statistically significant differences (P<0.05 or P<0.01) . The differences of ACT scores between the two groups were insignificant after treatment ( P>0.05) , but were significant ( P<0.05) 12 months later. (2) Within one year after treatment, the frequency of acute asthma attack was decreased in both groups (P<0.05 compared with that before treatment), and the treatment group had better effect on decreasing the attack frequency than the control group (P<0.05). (3) After treatment, the serum IgE, CD4+level, and CD4+/CD8+ ratio were decreased significantly, while CD8+ level was increased in the treatment group, and there were statistically significant differences ( P<0.05 compared with those before treatment). However, the differences of immune indexes in the control group were insignificant before and after treatment ( P>0.05). The improvement of immune indexes in the treatment group was superior to that in the control group ( P<0.05). Conclusion WXPR has good clinical efficacy in preventing and treating bronchial asthma. The mechanism is related with the inhibition of airway inflammation and improvement of immune function probably through regulating T lymphocyte subsets and lowering serum IgE level in asthma patients.

BACKGROUND:Matrix attachment region(MAR)are DNA elements that bound to the nuclear matrices after chromatin digested with restriction endonuclease.Plenty of studies have shown that MAR considered as initiaI point of DNA replication or transcription of regulatory gene.Thereby,construction of MAR expression vector can elevate the overall level of transgene expression,enhance stability of exogenous gene.as welI as increase frequency of stable transfectant cells.OBJECTIVE:To construction pLXSN-CAT recombinant retrovirus vector that containing chloramphenicol acetyltransferase(CAT)via cloning MAR sequence of human.and to explore the influence of MAR on the gene expression.METHODS:An open experiment was performed at the Department of Biochemistry and Molecular Biology.Xinxiang Medical College from September 2007 to December 2007 The PLXSN-CAT vector of CAT was constructed by the laboratory.TaqDNA polymerase,T_4 DNA ligase,DNA Marker,restriction enzyme BamH I,agarose gel DNA purification kit,as well as plasmid purification kit were purchased fromTakara Biotechn0Iogy(Dalian)Co.,Ltd.The sequence of csp-B MAR was amplified by polymerase chain reaction(PCR)method applied to human DNA.The fragment was inserted into retrovirus vector PLXSN-CAT plasmid.The recombinant plasmid was verified by double digestion and DNA sequencing.RESULTS AND CONCLUSION:The length of specific fragment applied by PCR was 931 bp,and the recombinant plasmid PLXSN-CAT-MAR presented two bands:5.9 kb and 931 bp using respective restriction enzymes BamH I The sequence of MAR was confirmed by blasting to Genbank(serial numobr:M6271 6).It suggested that MAR had been cloned into PLXSN-CATR vector correctly.The recombinant retrovirus vector PLXSN-MAR was successfully constructed.

Objective To investigate the expression of hsa-miR-144 in esophageal squamous cell carcinoma, and its relationship with clinicopathological features and prognosis. Methods Reverse transcriptase polymerase chain reaction (RT-PCR) method was used to detect the hsa-miR-144 in 46 cases of esophageal squamous cell carcinoma and adjacent normal tissue. The expression of hsa-miR-144 in esophageal squamous cell carcinoma and its difference in the clinicopatho?logical characteristics including gender, age, and tumor size were investigated. The relationship between the expression of hsa-miR-144 and prognosis of patients with esophageal squamous cell carcinoma was analyzed. Kaplan-Meier method and Log-rank test were used to analyse the differences in survival rates in different pathological characteristics. Results The ex?pression level of hsa-miR-144 was lower in esophageal squamous cell carcinoma 0.97(0.22-24.48)×10-6 than that of adjacent normal tissue 8.60(0.09-258.20)×10-6, the difference was statistically significant (Z=2.221, P<0.05). The expression level of hsa-miR-144 was higher in esophageal squamous cell carcinoma with no lymph node metastasis than that in esophageal squa?mous cell carcinoma with lymph node metastasis (Z=2.758,P<0.05), and the expression level decreased with the increase in the pathological staging (Z=7.737,P<0.05). There were no significant differences in the expression levels of hsa-miR-144 between different gender, age, tumor size, tumor location, tumor differentiation and tumor invasion depth (all P>0.05). There was no correlation between the expression of hsa-miR-144 and prognosis in patients with esophageal squamous cell carcino?ma (rs=0.031, P=0.839). In the survival rate, there was no statistic significance between high expressive of hsa-miR-144 group and low expressive group (P=0.828). The survival rate was lower in patients with lymph node metastasis than that of pa?tients without lymph node metastasis. The survival rates were lower in patients with relatively deep invasion and higher patho?logic stage (P<0.05). Conclusion The expression of hsa-miR-144 is down regulated in esophageal squamous cell carcino?ma, and which is associated with lymph node metastasis and pathological staging of esophageal carcinoma. It shows that hsa-miR-144 may serve as an anti-oncogene in the occurrence and development of esophageal squamous cell carcinoma.

Objective To evaluate the anti-HBV effect of hypericin from the cellular level and to preliminarily explore its po-tential drug target point.Methods Liver cell line HepG2.2.15 cells secreting HBV particles were selected as the experimental ob-jects.Hypericin served as the HY group,lamivudine was taken as 3TC group and deionized water as the blank control group.The cells were grouped and administrated.The HBV-DNA copy level was measured at72 h after medication by Southern blot and fluo-rescent quantitative PCR;the inhibition rate of HBsAg and HBeAg was detected by using ELISA assay;the pgRNA expression level was tested by using Northern blot and fluorescent quantitative PCR;Western blot and fluorescent quantitative PCR were adopted to detect the expression of regulatory factors including HNF3β,HNF4α,PPARαand RXRα.Results Compared to the blank control group,both hypericin and lamivudine had significant inhibiting effect on HBV DNA and expression level of HBsAg and HBeAg in HepG2.2.15 cells (P <0.05).Hypericin could significantly decrease the pgRNA expression compared with the blank control group (P <0.05),while lamivudine had no obvious change (P <0.05).Moreover,hypericin exhibited significant effects on the expression of HNF3βand regulatory factor HNF4αcompared with the blank control group and 3TC group(P <0.05).Conclusion Hypericin represents a strong anti-HBV effect,moreover could increase the negative regulatory factor HNF3βn expression and decreases the positive factor HNF4αexpression,prompting that its drug target point could be pgRNA.

Objective To identify the T cell epitopes from ovarian cancer associated anti-idiotypic antibody 6B11in order to explore the molecular basis of 6B11 induced cellular immune responses against ovarian cancer.Methods Potential human leukocyte antigen(HLA)A0201 ligands were predicted by using SYFPEITHI algorithm and tested by the T2 binding assay for screening of HLA-A2 binding peptides from 6B11 complimentary determining region(CDR).Cytotoxic T lymphocytes(CTL)to 6B11 or peptides were generated by 3 rounds of in vitro stimulation with 6B11 or peptide-pulsod dendritic cells(DC),and then tested by 51Cr-release assay to ascertain the CTL epitope of 6B11.Cell proliferation assay was performed by using 6B11 specific CTL as responder cells and peptide-pulsed DC as stimulators to ascertain the helper T lymphocyte(Th)epitope of 6B11.Cytokine assay and interferon-γ ELISPOT assay were performed to verify the CTL and Th epitope of 6B11 further.Results Light chain CDR3 peptide(VL CDR3)of 6B11 induced specific CTL to kill HLA-A2 and target antigen positive ovarian cancer cells,which could be blocked by anti human major histocompatibility complex(MHC)Ⅰ antibody.Heavy chain CDR3 peptide(VH CDR3)of 6B11 stimulated the proliferation of 6B11-primed CTL,which could be blocked mainly by anti human MHC-Ⅱ antibody,and further experiments showed that part of the VH CDR3 peptide-primed CTL killed K562 cells.Peptides in VL CDR3 and VH CDR3 were the CTL and Th epitope mimicking the original antigen of 6B11 respectively.Collaboration of 6B11 CTL and Th epitope,or 6B11 CTL epitope and keyhole limpet hemocyanin(KLH),the former was more powerful in inducing specific cellular immune responses against ovarian cancer.6B11 or corresponding CTL and Th epitope specific CIL secreted high levels of interleukin-2 (1630,1503 ng/L)and interferon-γ(5620,5421 ng/L),while basal level of interleukin-4 was detected (253,274 ng/L).ELISPOT assay confirmed the existence of specific interferon-γ secreting cells in 6811 or epitopes specific CTL(196/1×106 T cell,184/1×106 T cell).Conclusions VL CDR3 and VH CDR3 peptides of 6B11 are the CTL and Th epitopes mimicking original antigen which could duplicate the activity of intact 6B11 to induce the cellular immune responses against ovarian cancer.The results have significant theoretical and practical value in application of anti-idiotypic antibody ag anti tumor vaccine.The acquired CTL and Th epitopes as constituents of future multiple peptides against ovarian cancer could be used in peptide vaccine based ovarian cancer therapy.