Objective To compare the efficiency of detecting YMDD mutations by direct DNA sequencing and real time fluerescence PCR, and to explore the significance of the mutations of drug-resistance gene other than YMDD mutation. Methods 92 serum samples from 89 patients with chronic hepatitis B were collected and all the samples were detected by real-time PCR for YMDD mutation. HBV DNA was extracted from serum and was amplified by nest PCR to achieve HBV P gene RT region sequence. The PCR products were sequenced at both directions, and the sequencing results were contrasted through NTI program. The other 11 known drug-resistance mutation sites at the HBV RT region were also analyzed. Results Among the 37 samples with no YMDD mutation detected by real-time PCR, 33 samples were with M204M (without mutation), 1 sample with M204I and 3 samples with M204V by direct sequencing. Mutation and wild-type standard sequences were all coexisted in the 4 positive samples. There were 7 samples detected with ADV resistant mutation, accounting for 18.9% (7/37). Among the 55 samples with YMDD mutation detected by real-time PCR, 52 samples were detected by DNA sequencing, the accordance rate was 94.5% (52/55); 5 samples with ADV or ETV resistant mutation were detected, accounting for 9.1% (5/55). Conclusions Direct DNA sequencing is a high sensitive, repeatable method to detect drug-resistance mutation at RT region of HBV P gene. The result is well consistent with that attained by real-time PCR. Direct DNA sequencing can also detect various drug-resistance mutations as well as YMDD mutation, which is helpful to generally understand the nucleoside analogue resistant mutation and adopt more reasonable therapy projects against HBV.

Edema, as one of common clinical diseases, could be treated by taking medicines and adopting external therapies with traditional Chinese medicines (TCM). In recent years, there have been many clinical and basic studies concerning external therapies with TCM on edema Data showed that the external therapies are mostly composed of such purgating drugs as Rhei Radix et Rhizoma, Natrii Sulfas and Pharbitidis Semen, heat-clearing drug such as Phellodendri Chinensis Cortex and resuscitation-inducing drug such as Borneolum Syntheticum. The study showed that ingredients of external therapies did not pass through hilum and hepatic system, and thus avoided the first pass effect of livers. They enabled effective components of drugs to be rapidly absorbed through pores and skins, strengthened the effect against edema, shortened the treatment course, decreased side effects, and were convenient and inexpensive. External therapies with TCM could play unique advantages in inhibiting edema in the future clinical studies.
Animals ; Drugs, Chinese Herbal ; administration & dosage ; Edema ; drug therapy ; Humans

Objective To discover the operating problems through analysis of the financial situations of the rehabilitation institutions. Methods According to the different regions and different levels of rehabilitation institutions, 416 representative institutions from 26 provinc-es, municipalities and autonomous regions were surveyed. Results 329 effective questionnaires were recalled. 57.75%institutions relied on the superior departments in charge of funding, 51.09%institutions were in breakeven state. They were lack of sustainable development pow-er, and the capital expenditure was mainly used for staff salaries and equipment purchase. Conclusion To solve the shortage of funds in the development of rehabilitation institutions, we should establish a long-term mechanism of internal and external compensation.

Objective To construct prokaryotic expression vector of XTP3 gene and induce the expression of fusion protein in E.coli,and prepare XTP3-specific rabbit polyclonal antibody,detect the specificity of the antibody in hepatic carcinoma tissue and normal liver tissue.Methods DNA fragment of XTP3 amplified by PCR was inserted into pET-32a(+) to construct prokaryotic expression vector pET-32a(+)-XTP3.After identified by sequencing,pET-32a(+)-XTP3 was transformed into E.coli BL21 and induced with IPTG.After analyzed by SDS-PAGE and Western blotting,the induced expression product was purified and renatured by Ni+ affinity column chromatography.The purified protein was used to immunize New Zealand rabbits to gain polyclonal antibody,and the polyclonal antibody was then detected by ELISA,immunohistochemistry and Western blotting.Results Prokaryotic expression vector pET-32a(+)-XTP3 was successfully constructed,and the XTP3 fusion protein of about 52kD was highly expressed in E.coli.DS-PAGE showed that the protein product was mainly in inclusion body.The purified protein and polyclonal antibody were obtained successfully.It was manifested by ELISA that the titer of polyclonal antibody was over 1∶128 000.Immunohistochemistry showed that XTP3 antibody presented membrane-positive in hepatic carcinomous tissue.Conclusions The recombinant XTP3 protein and polyclonal antibody have been obtained successfully.These results lay a foundation for studying the immuneogenicity and bionomics of XTP3 protein.

Objective To analyze the variation in CTL epitope of hepatitis B virus (HBV) popular in China. Methods Amino acid sequences of 13 cytotoxic T lymphocyte (CTL) epitopes previously reported abroad were taken as reference sequences, and comparison was made draw between these reference sequences and both the 45 CTL-epitopic sequences obtained from the Genbank and also 5 CTL-epitopic sequences from native patients with chronic hepatitis B, using Vector NTI software. Results Sequence variations were observed frequently within several CTL eptitopes of HBV genotype B and C compared with the reference sequences. In the 13 analyzed CTL epitopes, 4 showed variation accounting over 70% in genotype B, and 2 have variance over 70% in occurrence in genotype C. Among these, C18-27 (FLPSDFFPSV), HBcAg-derived CTL epitope which was the most frequently studied previously, is showed in variation more frequently in HBV genotype B and C popular in China, reaching 71% and 97%, respectively. Conlusion Some CTL epitopic sequences of HBV genotype B and C popular in China have their own charactiristic difference from previously reported ones, which should be considered in the study of CTL response of Chinese subjects.

OBJECTIVE To investigate the distribution and the resistance rate of Shigella in Anhui Province to guide the choice of antibacterials. METHODS Ninety one strains of Shigella were cultured in Sep 2005.The groups were identified by biochemical and serologic tests.Susceptibility of 91 strains of Shigella in Anhui to various antibiotics was tested using standardized custom dilution MIC panels according to CLSI(2005) guidelines. RESULTS There were 57 strains of Shigella flexneri,31 strains of S.sonnei and 2 strains ofS.boydii among 91 strains of Shigella.The resistance rates of Shigella to cefoperazone/sulbactam and piperacillin/tazobactam were remarkably lower than to other third generation cephalosporins.The susceptible rates to carbopenems were 100%.The resistance rates to ciprofloxacin lactate and pazufloxacin mesilate were 27.47% and 32.97%,respectively. CONCLUSIONS There is a certain resistance rate of the Shigella to fluoroquinolones and the third generation cephalosporins.More attention should be paid to the surveillance and control of such resistance.

Objective:To explore the relationship between YKL-40 and the proliferation of breast cancer and its mechanism.Methods:The expression of YKL-40 in breast cancer MCF-7 cells was detected by immunofluorescence assay.Fluorescence microscope was used to observe the conversion efficiency,and Real-time PCR was used to screen the most effective YKL-40 siRNA.Expression levels of PI3K,P-PI3K,AKT,P-AKT of the PI3K/AKT pathway associated proteins was test by Western blot.At the same time,MTT and flow cytometry were validated by YKL-40 siRNA treatment of human breast cancer MCF-7 ceils,the differences of 24h,48h and 72h groups of cell proliferation ability and cell cycle.Result:MCF-7 cell express YKL-40 protein,mainly located in the cytoplasm.Real-time PCR show that siRNA01,siRNA02,siRNA03 compared with NC group YKL-40 gene silencing effect is remarkable.Among of them the strongest silencing effect is siRNA02 (P＜0.01),Western blot show the experimental group than the control group,Total PI3K and AKT remain unchanged while P-PI3K,P-AKT expression decreased (P＜0.05).In the experimental group,the number of G1 cells in the control group was increased (P＜0.01),while the S phase cells decreased (P＜0.01).MTT results showed that the experimental group compared with the control group,the proliferation ability is decreased(P＜0.01).Conclusions This study suggests that YKL-40 can be used as the upstream regulatory factor of PI3K/AKT signaling pathway and affect the process of cell cycle in breast cancer,and then regulate the proliferation of breast cancer,YKL-40 may be a crucial target for the treatment of breast cancer.

Aim To research the effects of total flavones of rhododendra(TFR)on transient receptor potential vanilloid receptor 4(TRPV4)in cerebral basilar arteries(CBA)of rats subjected to ischemia/reperfusion(IR)injury.Methods The model of total brain IR was established by four-artery occlusion(4-VO)method in rats.Arterial pressure perfusion and cell membrane potential recording methods were used for surveying the dilatation and hyperpolarization of TFR and ruthenium red(RR,an inhibitor of TRPV4)in the KCl-preconstricted CBA ex vivo in rats subjected to IR.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot were utilized to investigate the TRPV4 mRNA and protein expressions of TFR and RR in cerebrovascular endothelial cells of CBA in vivo in rats subjected to IR.Results 11～2 700 mg·L-1 TFR significantly induced concentration-dependent hyperpolarization and dilatation in the KCl-preconstricted CBA in rats subjected to IR.TFR still produced degenerative hyperpolarization and dilatation by removal of endothelium in CBA,which was remarkably attenuated as compared with endothelium-intact group(P<0.01).After removal of NO and PGI2 vasodilatation,TFR obviously elicited the hyperpolarization and dilatation that were further decreased by RR(an inhibitor of TRPV4)in IR CBAs.TFR pretreatment apparently increased the level of TRPV4 mRNA and protein expressions in IR CBAs.These effects were restrained by RR,an inhibitor of TRPV4.Conclusions TFR could mediate endothelium-dependent and endothelium-independent effects.The endothelium-derived dilatation may be related to the increase of endothelium activity and endothelium-derived hyperpolarizing factor(EDHF)generation and release that have been promoted by TFR,and secondarily activating TRPV4,which results in Ca2+ inflow and subsequent hyperpolarization of vascular smooth muscle cell membrane and vasorelaxation.

Objective To investigate the therapeutic effect of the hemilaminectomy approach in intraspinal tumor with the help of intraoperative ultrasound,high-speed drill and ultrasound knife.Methods Clinical data of 35 cases with the intraspinal tumor were analyzed retrospectively.All the patients were treated by hemilaminectomy approach,the position of the lesions were fixed by preoperative X-ray and intraoperative ultrasound,root of the spinous process were removed in order to achieve adequate visual field by high-speed drill and ultrasound knife.Electrophysiologic monitoring were used if the lesions were in the spinal cord.Results Total surgical resection was achieved in 32 cases,gross total resection was in 3 cases.Twenty-eight of the 35 cases were followed up.The symptom alteration of the first period ( 1 - 3 months postoperative):improved in 25 cases,no changed in 2 cases,aggravated in 1 case.The symptom alteration of the second period (6-24 months postoperative):no changed in all cases,no stability change of the vertebral column had been found.Conclusions Followed with the development of the electrophysiologic monitoring,high-speed drill,intraoperative ultrasound,the indication of the hemilaminectomy and have no influence of the spinal stability.However,to spinal meningioma which have a wide base,the hemilaminectomy approach may not adapt.

The secondary development of traditional Chinese medicines (TCMs) is an important content of TCM modernization process, as well as an important path for developing new TCM drugs. Under the guidance of the system theory, in response to the lack of the overall guideline and practical methods for the secondary development of TCMs at present, we introduced the overall thought of the secondary development of major TCM varieties, as well as the roles and contents of clinical research, pharmacology and pharmaceutics in the process of the secondary development of major TCM varieties, so as to provide systematic strategies and methods for the development of major TCM varieties.
Biomedical Research ; Drug Discovery ; methods ; Humans ; Medicine, Chinese Traditional ; methods ; Pharmacology