Objective: To construct a recombinant adenovirus (abbreviated as ET-M9-PEX) containing MMP-9 signal peptide and noncatalytic carboxyl-terminal hemopexin domain of human MMP-2, and to use the constructed adenovirus as a drug bioreactor in vivo to enhance the expression of an anti-angiogenesis factor for treatment of tumor by a gene therapy strategy. Methods: Adenovirus vector containing M9-PEX gene was constructed by PCR and gene recombination, and was packaged and amplified in L293 cells to obtain ET-M9-PEX recombinant adenovirus with infective ability. The expression and secretion of PEX in ET-M9-PEX-infected cells were detected by Western-blotting and immunofluorescent staining. The inhibitory effect of ET-M9-PEX-conditioned medium on EC cells proliferation was detected by growth curve and its inhibitory effects on angiogenesis and tumor growth were determined by chicken chorioallantoic membrane (CAM) assay in vivo. Results:ET-M9-PEX was successfully constructed and the expression and secretion of PEX in ET-M9-PEX-infected cells were verified. The ET-M9-PEX conditioned medium significantly inhibited the proliferating rate of EC cells. The tumor weights from ET-M9-PEX-infected PG cells in CAM and gradeⅠvessel number were reduced by 57.57%(P

Objective The aim of this study was to prepare anti-snail polyclonal antibody and make it widely useful in snail detection. Methods The DNA fragment encoding human full length 264 amino acid of snail was obtained by PCR from cDNA library of human umbilical vein epithelial cells and was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST).The GST-snail fusion protein was expressed by E.coli BL21 after IPTG induction and purified from total proteins of BL21 transformed by the recombinant plasmid pGEX-4T-1/snail.The New Zealand rabbit was immunized with the purified fusion protein to prepare polyclonal antiserum.The antiserum was identified by western blotting and immunofluorescent staining. Results The prokaryotic expression plasmid pGEX-4T-1/ snail was successfully constructed,and the fusion protein GST-snail was expressed efficiently.The polyclonal antibody raised in the rabbit could react specifically with snail in human cells.Conclusion The snail antiserum was of good purity with high titer and specificity which could satisfy the requirement for studying immuno-analysis on snail protein.

Objective: To further understand the roles of gap junctional intercellular communication in carcinogenesis and progression of human lung cancer. Methods: The heterologous communication was characterized among human lung epithelial cells HLEC, human lung fibroblasts HLF, human lung giant carcinoma PG cells and its Cx43 transfectants PG/C4 by using a preloading assay. Cx43 immunofluorescent staining and Northern blot were performed to examine Cx43 gene expression. Results: Although both human lung firoblasts HLF and epithelial cells HLEC expressed Cx43 and had very strong homologous communication, HLEC cells were unable to communicate with HLF cells. The human lung carcinoma cell line PG was defective of both homologous communication, and heterologous communication with its epithelial origin HLEC. Transfection of Cx43 into PG cells, which rescued PG cells' homologous communication and enhanced heterologous communication with HLF cells, could restore heterologous coupling with HLEC cells. Conclusion: Selective heterologous communication exists among different kinds of human lung cells. when human lung carcinoma cells lost coupling with its epithelial origin, Cx43 gene expression is not enough to establish heterologous communication between them.

Objective To inspect the quality of Fructus Aurantii Immaturus formula granule. Methods Choose three batch Fructus Aurantii Immaturus to prepare formula granule and decoction. Zobax Extend C18 column was used with acetonitrile-water-phosphoric acid-sodium lauryl sulphate (12∶87∶1∶0.2) as the mobile phase, the detection wavelength was 275 nm. Result There were no differences in elution peak quantity and peak position in the HPLC spectrum of Fructus Aurantii Immaturus formula granule with corresponding cut crude drug and decoction. The relative peak area in the corresponding peak of formula granule had no significant difference with decoction, but had certain differences with cut crude drug. Conclusion HPLC spectrum showed that Fructus Aurantii Immaturus formula granule and decoction had high similarity.

Objective To detect the expression level of RKIP in human lung cancer cell lines and to investigate the relationships between RKIP and cell metastasis. Methods The expression level of RKIP in human lung cancer cell lines was analyzed by RT-PCR. To construct and express human RKIP fusion protein and to immunize New Zealand rabbit with purified fusion protein to prepare polyclonal antiserum.The antiserum was titered by ELISA. The specifcity of antiserum and expression level of RKIP in human lung carcinoma cell lines was analyzed by western blotting. Results The fusion protein was successfully expressed. The prepared polyclonal antibody reacted specifically with RKIP in human cells. Downregulation of RKIP was detected in three lung cancer cell lines with metastasis, and it is more significant in A549 with higher metastasis.Conclusion The RKIP antiserum with high titer and good specificity could meet the requirement for studying on RKIP. Downregulation of RKIP has correlation with human lung carcinoma cell metastasis.

Objective To investigate the effects of percutaneous coronary intervention(PCI) and conventional drugs on autonomic nervous system and C-reactive protein (CRP)in the patients with unstable angina pectoris combined by QRS complex fragment. Methods A total of 60 patients aged (46.2± 10.3) years with unstable angina combined by QRS complex fragment were randomly divided into drug therapy group and PCI group (n=30 for each group).The changes of heart rate variability (HRV) including SDNN,SDANN,rMSSD,PNN50,HF and LF,heart rate turbulence(HRT) such as TO and TS,and CRP were measured before and 1 month after treatment. Results The values of SDNN[(88.2±20.6)ms vs.(122.5 ± 15.5)ms; (86.9± 23.4)ms vs.( 106.7± 18.8)ms],SDANN [(76.2±9.3)ms vs.(105.3±5.2)ms; (74.3±10.4)ms vs.(89.8±7.6)ms],rMSSD[(18.6±7.9)ms vs.(49.3± 4.3)ms; (19.3± 7.4)ms vs.(29.4± 5.2)ms],PNN50 [(5.5± 2.8)％ vs.(9.1 ±1.8)％; (5.3±2.1)％ vs.(7.2±3.2)％],HF[(219.4±131.6) Hz vs.(292.5±125.5) Hz;(217.2±133.2) Hz vs.(213.2±120.2 ) Hz] and LF[(459.6±135.2) Hz vs.(345.1±175.1) Hz ;(445.8± 144.3) Hz vs.(396.1 ± 182.3) Hz] were improved after treatment as compared with pretreatment in PCI group (t=9.4,15.69,8.37,4.68,3.26,3.57,P＜0.01 or 0.05) and in drug therapy group (t=7.3,12.36,6.98,2.94,4.89,5.01,P＜0.01 or 0.05),respectively.The changes of above indexes were more remarkable in PCI group than in drug therapy group(t=8.90,13.75,7.52,3.27,4.21,4.01,P＜0.01 or 0.05).The values of turbulence onset(TO) [(0.45±0.44)％ vs.(0.16±0.20)％,t=15.63,P＜0.01; (0.49±0.38)％ vs.(0.32±0.26)％,t=16.78,P=0.001] and turbulence slope (TS) [(2.12±0.13)ms/RR vs.(2.98±0.25)ms/RR,t=14.36,P=0.001; (2.15±0.19)ms/RR vs.(2.51±0.11)ms/RR,t=19.52,P=0.001] and CRP [(5.74±2.46)mg/L vs.(2.61±1.22)mg/L,t=12.49,P=0.001; (5.81±2.35)mg/Lvs.(3.56±1.43)mg/L,t=9.76,P=0.01] were also improved after treatment as compared with before treatment in PCI and drug therapy groups,respectively,and the the changes in TO (t=15.95,P＜0.001),TS (t=18.13,P=0.001) and CRP(t=10.73,P=0.001) were more obvious in PCI group than in medicine group. Conclusions PCI may obviously reduce inflammatory response,provide more myocardial blood supply and improve autonomic nervous function in the patients with unstable angina pectoris combined by QRS complex fragment.

Objective To construct a lentivirus vector for RNA interference targeting myosin Va gene and to observe its effect on motility and migration of human pulmonary giant cell carcinoma PG cells. Methods Based on the efficient target sequence for myosin Va RNAi, two pairs of oligo DNA containing myosin Va RNAi target sequence or scramble sequence were synthesized and inserted into pSuper vector, followed by sequence analysis. The expressing cassette H1 promoter-shM5A/shCON from the recombinant pSuper plasmid was then transferred to the lentivirus vector plenti4, and the recombinant lentivirus was packaged. PG cells were transduced with the packaged lentivirus and the positive cells were screened by zeocin selection. RT-PCR was performed to determine the myosin Va RNAi efficiency in zeocin-resistant PG cells, and wounding assay and Boyden chamber assay were utilized to examine the capabilities of motility and migration in myosin Va RNAi PG cells. Results Restriction enzyme digestion and sequencing confirmed the successful construction of the lentivirus vector containing myosin Va RNAi target or scramble sequence. RT-PCR result showed that myosin Va mRNA levels were remarkably reduced in lentivirus-based myosin Va RNAi PG cells. The abilities of motility and migration were also significantly inhibited in lentivirus-based myosin Va RNAi PG cells, as demonstrated in wounding assay and Boyden chamber assay.Conclusion Myosin Va RNAi lentivirus vector was successfully constructed and efficiently repressed myosin Va expression in PG cells. Repression of myosin Va by RNAi led to the inhibition of PG cells motility and migration, indicating that there might exist correlation between the expression of myosin Va and cancer progression.

Background and purpose:Micro-environmental hypoxia is a common phenomenon in most human solid tumors,and this investigation is done to observe the expression of HIF-1? and chemo-resistance-associated genes in human colon cancer cell line under hypoxic micro-environment in vitro,and study the influence of micro-environmental hypoxia on chemo-resistance and the possible mechanisms in human colon cancer.Methods:Human colon cancer cell line SW620 was cultured under hypoxia for 12,24,48 hr,with normoxia as control.Then the expression of HIF-1? and chemo-resistance-associated genes mdr1/P-Gp、LRP were investigated by RT-PCR and western-blot.Results:With prolongation of the hypoxic time,the mRNA expressions of HIF-1? and LRP remained at the same level,but the mRNA expressions of mdr1 showed a time-dependent increase(P

OBJECTIVE: To compare the contents of main chemical components in prescription granules and decoction of Fructus aurantii immaturus. METHODS: 3 batches of cut crude drugs of Fructus aurantii immaturus were selected to prepare the granules and decoction. The assaying of water soluble extractive was conducted according to the specification of China Pharmacopeia. The assaying of hesperidin and synephrine was conducted by HPLC. RESULTS: There was no obvious difference between water soluble extractive of granules and that of decoction, while the contents of hesperidin and synephrine were higher in the prescription granules than in the decoction. CONCLUSIONS: The results provide supporting basis for the labeled amount of prescription granule of Fructus aurantii immaturus.

: Objective To develop a rehabilitation device and software game, and provide the system for evaluation and training of anterior cruciate ligament (ACL) reconstruction.MethodsThe development and clinical application of a biofeedback knee joint movement rehabilitation system for ACL reconstruction were very important.ResultsBIOKS readings reproduce Zebris 3D motion analysis system measurements reliably between 0° and 150° ( P>0.05). Significant knee joint movement training effect ( P<0.05 ) in JPS assessment. However, there were no differences ( P≥0.05) between SST and KAS assessments.ConclusionThe improvement of joint position sense and body centroid sway under single leg support conditions.