Objective: Extracting ephedrine and berberine from the water extract of Ephedra Herba and Rhizoma Coptidis. Methods: Modified PVA was used as separating membrane and separated alkaloids. Results: A colorless ephedrine aqueous solution was obtained when extraction time was less than 8 hours; while a three constitution solution was obtained when time of extraction is over 8 hours. So is Rhizoma Coptidis. The membrane is hardly fouled. Conclusion: Purity of alkaloids from Herba Ephedra and Rhizoma Coptidis by membrane separation is Superior to that by alcoholic sediment but amount of alkaloids is reverse.

Objective To investigate the efficacy and prognostic factors in patients with stage Ⅰ b and Ⅱ a cervical carcinoma by radical radiotherapy.Methods Between January 1999 and January 2012,108 patients with stage Ⅰ b and Ⅱ a cervical carcinoma received radical radiotherapy in Cancer Hospital,Chinese Academy of Medical Sciences were included and analyzed retrospectively.Patients of stage Ⅰ b1,Ⅰ b2,Ⅱa1 and Ⅱa2 were 18 (16.7％,18/108),38 (35.2％,38/108),33 (30.6％,33/108) and 19 (17.6％,19/108),respectively.Results The 5-year overall survival rate was 76.2％ and the 5-year disease free survival rate was 75.6％.Totally 25 (23.1％,25/108) patients developed recurrent disease,16 of them (64％,16/25) had local recurrences,6 (24％,6/25) had distant metastases and 3 cases had both local recurrence and distant metastases.Among patients with recurrent disease,23 died and 2 survive with tumor.Totally 24 patients died,23 of them died due to tumor recurrence and the other one died of other reason.The univariate analysis showed that,lymph node metastasis,squamous cell carcinoma antigen (SCC) levels before treatment,SCC levels after treatment 1 month had relation with overall survival time in patients with stage Ⅰ b and Ⅱ a cervical carcinoma (all P ＜ 0.05).The multivariate analysis showed that,lymph node metastasis and SCC levels after treatment 1 month were the independent prognostic factors for overall survival time for the cervical squamous cell carcinoma (OR =2.5,4.4 ; all P ＜ 0.05).Conclusions By means of radical radiotherapy,stage Ⅰ b and Ⅱ a cervical carcinoma patients with lymph node metastasis and SCC levels≥ 1.5 mg/L after treatment one month have poor prognosis.While,stage Ⅰ b and Ⅱ a patients with concurrent chemoradiotherapy after neoadjuvant chemotherapy did not affect the prognosis.The 5-year survival rate with concurrent chemoradiotherapy was higher than that of radiotherapy.

HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerase gene to express the target protein. HBV polymerase expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

The effects of PEG10 on hydrogen peroxide (H(2)O(2))-induced apoptosis in human normal liver cell line L0(2) were investigated. The PEG10 gene was transfected into L0(2) cells by lipofectamine, the positive clone was screened by G418 and defined as L0(2)/PEG10, while the cell transfected with empty expression vector (pEGFP-N1) was defined as L0(2)/vector. L0(2)/vector and parental L0(2) cells served as control. RT-PCR and Western blotting were employed to detect the expression of target genes. H(2)O(2) (50-400 mmol/L) was administered to induce the apoptosis of L0(2) cells. Cells viability was measured by MTT and the morphological changes of apoptotic cells were determined by fluorescence microscopy using hoechst33342 nuclei staining. DNA fragmentation was observed by agarose gel electrophoresis. PEG10 mRNA and protein levels in L0(2)/PEG10 cells were significantly increased as compared with those in the control cells. After treatment with 400 mmol/L H(2)O(2) for 24 h, the cellular growth inhibition rate of L0(2)/PEG10 cells was significantly lower (58.2%) than that of L0(2) (92.5%) and L0(2)/vector (88%). Distinct morphological changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were not observed in L0(2)/PEG10. Ladder-like DNA fragmentation in a dose-dependent manner was observed in both L0(2) and L0(2)/vector cell lines, but not in L0(2)/PEG10. PEG10 over-expression significantly inhibited cytotoxicity induced by H2O2 on human normal liver cell line L0(2) by antagonizing H(2)O(2)-induced apoptosis.

Objective To probe into the advantages and disadvantages of intravenous chemotherapy and intraperitoneal chemotherapy for advanced epithelial ovarian cancer.Methods All of the 226 patients with advanced epithelial ovarian cancer were treated by maximum cytoreductive surgery or non-effective cytoreductive surgery and received 6-8 courses of postoperative regular chemotherapy(chemotherapy regimens,TP:taxol and cis-platinum or carboplatinum;PC:cis-platinum and cyclophosphamide;PAC:cis platinum and adriamycin and cyclophosphamide)during Jan 1998-Jan 2006.We systematically compared the characteristics of patients in intraperitoneal chemotherapy(IPC)group and intravenous chemotherapy(IVC)group.We measured the incidence rate of the response,side-effects,the recurrence time of intraperitoneal tumor and survival time of the two groups respectively.ResultsFor the first phase after operation(three courses of treatment),the response rate of two groups were 75.8％and 52.8％respectively.For the response rate of IPC was higher than that of IVC(P＜0.01).The second phase after operation(all courses finished),the response rate of two groups were 93.9％and 87.7％,respectively(P＞0.05).After maximum cytoreductive surgery,the recurrence rate of IPC and IVC were 47.0％and 59.4％,respectively(P＞0.05).After non-effective cytoreductive surgery of IPC and IVC groups,the recurrence rates were 84.8％and 86.2％,respectively(P＞0.05).The recurrence time of intraperitoneal tumor of IPC and IVC groups were 24 and 18 months,respectively(P=0.001).The overall survival time ofgroups IPC and IVC were 32 and 30 months(P=0.188).There were some differences in the side-effect between IPC and IVC.The rates of chemotherapeutic phlebitis of IPC and IVC were 34.0％and 10.8％ respectively(P＜0.01).The rates of serious gastrointestinal reaction of IPC and IVC were 33.8％and 25.8％,respectively(P=0.236).There was no significant difference in bone marrow depression,intestinal adhesion and intestinal obstruction.ConclusionsIPC can extend the disease progression free survival than IVC,without increasing overall survival period.IPC can also reduce the side-effect of chemotherapeutic phlebitis.However,IPC is used limitedly,and can not substitute for IVC.Combination of IPC with IVC may enhance their effectiveness and reduce the side-effects.

Objective To analyze the clinicopathologic characteristics,treatment and prognostic factors in malignant transformation of mature cystic teratoma(MCT)of ovary.Methods The clinical data of 44 patients with MCT from January 1961 to June 2009 were reviewed.Results The median age of the 44 patients was 48 years(range,16-84 years).Mean tumor size was(16 ±6)cm.Thirty-two cases were diagnosed squamous cell carcinoma(73％,32/44),and 5 of them with the elevated level of serumal squamous cell antigen(SCC-Ag).Three of 37 cases(8％,3/37)were identified with malignant transformation in image examinations.Rapid frozen section examination and multiple-location biopsy were performed in 8 cases,and 5 of them were detected with malignant diseases.Twenty-two patients with disease confined within the unilateral ovary(10 with intact capsule,and 12 with ruptured capsule).Diseases extended extra ovaries in the others 22 patients.The median cumulative overall survivals were 126 and 10 months,respectively.The difference between the two groups was significant(P ＜ 0.01).Twenty-seven patients had no residual tumor after primary surgery.The median cumulative overall survivals between the patients with and without residual tumor were 10 and 84 months respectively,and there were significant difference between two groups(P ＜ 0.01).Seven selected patients with malignant disease confined within unilateral ovary underwent fertility-sparing surgery,and 2 cases of them had successful pregnancies and delivery,while other 4 cases with ruptured capsule recurred.Conclusions The most common pathology type of malignant transformation in mature cystic teratoma of the ovary is squamous cell carcinoma.Comprehensive pre-operation image examination and tumor marker level detection might be of great help in diagnosis.Tumor extension extraovary and residual tumor after surgery are the most significant poor prognostic factors.Early stage patient with ruptured capsule should be very discreet to choose fertility-sparing surgery.

Objective To explore the effective method in treatment of traumatic intracranial hematoma.Methods The measures and effects of traumatic intracranial hematoma treated by combined treatment of traditional Chinese medicine and western medicine were retorspectively analyzed. 62 cases with traumatic intracranial hematoma were taken controlled methods of clinical research: the control group had 30 patients ( received Western medicine),and experimental group had 32 cases ( received Western medicine treatment combined with Chinese medicine Qingkailing or/and Ligustrazine at the same time ), therapeutic effect evaluation was mede after 28 days.Results Clinical results of the experimental group was superior than the control group ( P ＜ 0.05 ); and complications were fewer (P ＜ 0.05). Conclusion Integration of Chinese and western medicine could improve the efficacy of traumatic intracranial hematoma.

BACKGROUND: Human keratinocyte growth factor-2 (hKGF-2) has extensive physiological functions, which plays an important role in embryonic development, tissue-repairing, nervous regeneration, vascularization and development of tumor.OBJECTIVE: To clone hKGF-2 gene, obtain the expression of hKGF-2 in Escherichia coli(E.coli) and determine its bioactivity, so as to provide experimental basis for further investigation.DESIGN: Open experiment.SETTING: Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.MATERIALS: The experiment was conducted in State Key Laboratory of Viral Genetic Engineering, Institute for Viral Disease Control and Prevention of Chinese Center for Disease Control and Prevention. The temperature control expression vector pBV220 was constructed by State Key Laboratory of Viral Genetic Engineering; EcoR Ⅰ , BamH Ⅰ , T4 DNA ligase (Promega Co., Ltd.); The specific polymerase chain reaction (PCR) of hKGF-2 (Manufactured by Shanghai Boya Biotechnology Co., Ltd.); Heparin-Sepharose CL-6B (Pharmacia Company); PCR rapid purification kit,Trizol kits for total RNA extract, Kits for RT-PCR (GIBCO Co., Ltd.); Kits for rapid extraction of plasmid DNA (Boda Company); BL-21-codon plus compent cells (Stratagene Co., Ltd.).METHODS: High-expression strain BL-21 codon plus competent cells was used to express and purify initially recombinant hKGF-2 protein, and its activity was detected. RT-PCR was adopted to obtain hKGF-2 cDNA from lung tissues of naturally aborted fetus and clone it into pBV220 carri er plasmid. The hKGF-2 protein expressed in BL-21 codon plus competent cells of E.coli. Affinity chromatography and ion exchange chromatography were applied in isolation and purification, and the bioactivity of expression protein was determined in cell proliferation test.MAIN OUTCOME MEASURES: The length and sequence of cDNA segment in hKGF-2, the expression of hKGF-2 gene inE.coli and the purification of hKGF-2 activity.RESULTS: The segment of hKGF-2 cNDA was about 500 bp, and hKGF-2 protein highly expressed in BL-21, which had soluble expression in the supernatant. SDS-PAGE showed that the relative molecular mass was about 20000, and hKGF-2 protein could significantly promote the mitotic activity of NIH3T3 cells. The A value (490 nm) of hKGF-2 in the 1 μg/L, 5 μg/L and 10 μg/L groupswere higher than that in the blank control group, and the differences were significant (which were 0.174±0.022,0.220±0.029,0.306±0.050,0.066±0.004 respectively,P ＜ 0.001).CONCLUSION: hKGF-2 gene is successfully cloned, which highly expresses in BL-21 of the E.coli. Purified hKGF-2 protein can stimulate the proliferation of NIH3T3 cells and significantly promote its mitotic activity.

Objective To investigate the relationship between single nucleotide polymorphism (SNP) of Fas ligand (FasL) and fulminant hepatitis B in Han Chinese. Methods HBV infected subjects were enrolled in this case-control study, including 233 cases of inactive HBsAg carrier, 68 patients with fulminant hepatitis B,100 cases of spontaneous hepatitis B clearance, 102 patients with hepatitis B virus (HBV) related cirrhosis and 112 patients with HBV related primary hepatocellular carcinoma. The blood samples and clinical data were collected. FasL-844T/C polymorphisms of enrolled subjects were examined by TaqMan real time fluorescent genotyping polymerase chain reaction (RT-PCR). A adjusted odds ratios (OR)and 95% confidence intervals (CI)were calculated using the Logistic regression model. Results After adjusting the factors of gender and age, binary Logistic regression analyses indicated that the genotype frequencies of FasL-844 CC,CT,TT in inactive HBsAg carriers were 50. 64% ,39. 91% and 9. 44% respectively, and those in cases of fulminant hepatitis B were 79. 41%, 17. 65% and 2. 94%, respectively. The analysis also revealed that FasL-844CC genotype in inactive HBsAg carriers was high risk factor of developing fulminant hepatitis B (OR =4. 729,95%CI:0. 510 - 21. 282,P = 0. 043), while there were no statistic significances in other cases (P>0. 05). Conclusion The inactive HBsAg carriers harboring FasL-844CC may have greater susceptibility to fulminant hepatitis B, which need arouse high attention.

To study the influence of recombinant endostatin on angiogenesis and tumor growth of mice H22 hepatoma, tumor models were constructed by injecting H22 hepatoma cells into the leg muscle of mice. Recombinant endostatin was produced by gene engineering in E. coli. The recombinant protein was injected subcutaneously to treat transplanted hepatoma faraway. The weight of tumors was measured, and the changes of necrosis of tumor cells and vessel density were observed by immunohistochemistry. The results suggested that the growth of hepatoma models transplanted in the muscle of legs was suppressed by recombinant endostatin. The density of vascularity was decreased, but the necrosis of tumor cells increased. The inhibitory effect of recombinant endostatin on angiogenesis and tumor growth of hepatoma was not affected after chemotherapy.
Angiogenesis Inhibitors ; pharmacology ; Animals ; Antineoplastic Agents ; pharmacology ; Endostatins ; biosynthesis ; genetics ; pharmacology ; Escherichia coli ; genetics ; Liver Neoplasms, Experimental ; blood supply ; pathology ; Male ; Mice ; Neoplasm Transplantation ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology