Although autoallergic neural transplantation Is a gold standard to repair neurologic defect, nerve tissue engineering becomes an ideal replacement due to a limited collection of nerve. Nerve tissue-engineering release-controlled system promotes axonal regeneration via a scaffold to slowly release nerve growth factor and to create a suitable microenvironment for nerve growth. There are various materials and methods for creating nerve tissue-engineering release-controlled system; therefore, choosing a good material and a good method to control nerve growth factor and to cause excellent repairing effect are hot topics for researching nerve tissue-engineering release-controlled system. The aim of this review is to introduce the new methods and technologies applied in the delivery system of nerve growth factors in recent years. This review also attempts to classify the strategies of drug delivery of nerve growth factor in a new way.

Objective To investigate the factors relevant to the vascular calcification in uiemic patients.Methods Eighty-five uiemic patients were enrolled in this study.The levels of fetuin-A,serum calcium,serum phosphorus,C-reactive protein and other parameters related to calcification were examined.B-ultrasound was used to detect carotid plaques.Results The Fetuin-A levels in patients with vascular calcification were significantly lower than those with non-vascular calcification[(2.34±0.95) μg/L vs (3.79±1.19) μg/L,t=5.94,P＜0.01],but serum calcium,serum phosphorus and C-reactive protein were higher than those non-vascular calcification [serum phosphorus (1.97±0.23) mmol/L vs (1.80±0.33) mmol/L,t=2.05,P＜0.05;calcium and phosphorus product (50.04±6.61) mg~2/dl~2 vs (44.84±9.75) mg~2/dl~2,t=2.05,P＜0.05;C-reactive protein (33.45±25.11)mmol/L vs (20.65±13.43) mmol/L,t=2.03,P＜0.05].Linear correlation analysis indicated that low fetuin-A level was correlated with C-reactive protein (r=-0.43,P＜0.01),calcium-phosphorus product (r=-0.32,P＜0.01),serum albumin concentration (r=0.37,P＜0.05) and phosphorus level (r=-0.36,P＜0.05).Conclusions The risk factors relevant to the vascular calcification are high serum phosphorus,calcium and phosphorus product and the micro-inflammatory status in uiemic patients.Vascular calcification is also correlated with low fetuinA level,adding exogenous Fetuin-A may become an effective means in preventing vascular calcification.

Objective To clone amastin coding genes from different strains of Leishmania parasites. Methods Using amastin cDNA sequence as the reference, dbEST data base established by National Center Biotechnology Information (NCBI), USA, was searched by BLAST tool. A 309 bp DNA fragment of Leishmania major was found and used as the probe for the screening of a DNA library. The amastin gene of Leishmania major Abdou was cloned and sequenced. Specific primers were designed and amastin genes for Leishmania mexicana WR972, Leishmania brizeliensis and Leishmania amazonensis joseph were amplified by polymerase chain reaction. Results The amastin genes from 4 strains of Leishmania parasites were cloned and sequenced. It was found that all 4 amastin genes contained unique open reading frame of 552 bp and encoded amastin protein of 183 amino acid residues. Conclusion The amastin genes of 4 strains of Leishmania parasites were successfully cloned.

To construct a cDNA subtractive library of genes transactivated by hepatitis C virus core protein with suppression subtractive hybridization technique. mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-)-core and pcDNA3 1(-) empty vector,respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNA were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, and then it was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain JM109. The cDNA were sequenced and analyzed in GenBank with Blast search after PCR. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR product showed that 213 clones contained 100~ 1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes before. The full length sequences were obtained with bioinformatics method,which had been accepted by GenBank. It suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein.

Objective To construct a cDNA subtractive library of genes transactivated by c-terminally truncated middle surface protein of hepatitis B virus(MHBs t) with suppression subtractive hybridization technique for cloning genes associated with transactivation. Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-Mt167 and pcDNA3.1(-) empty vectors, respectively, then cDNA was synthesized. After restriction enzyme Rsa I digestion, small-size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR and then was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. Results The subtractive library of genes transactivated by MHBs t was constructed successfully. The amplified library contained 94 positive clones. Colony PCR showed that these clones contained 200-800bp inserts. Sequence analysis was performed in 50 clones,and the full length sequences were obtained with bioinformatics method. 23 coding sequences were obtained in total, which consisted of 19 known and 4 unknown ones.Conclusions The obtained sequences may be target genes transactivated by MHBs t, among which some genes coding proteins may involve in cell cycle regulation, immune response and tumour genesis.

In order to screen genes transregulated by core protein of hepatitis C virus, cDNA microarray technology was employed to detect the gene expression change between HepG2 cells transfected with pcDNA3 1(-) core and the empty vector, respectively. Among 1152 genes, there were 95 genes with different expressions, of which 45 genes were upregulated and 50 genes were downregulated in HepG2 cells transfected with core protein expression plasmid. These genes transregulated by HCV core protein included human genes encoding proteins involved in cell proliferation, differentiation, apoptosis, signal transduction and immune regulation. Therefore, the results provided some new clues for further clarifying the molecular biology mechanism of pathogenesis and tumorigenesis of HCV core protein

Hepatitis C virus (HCV) non structure 3 (NS3) protein and hepatitis B virus (HBV) X protein expressing plasmids were constructed with the vector pcDNA3 1(-). The plasmids were transfected into HepG2 cells and the viral proteins expressed in HepG2 cells were identified using Western blotting methods. Then the two recombined plasmids were transfected into HepG2 cells and were cotransfected into HepG2 cells with reporter plasmid pCAT3 promoter. The activity of CAT enzyme was detected by a CAT ELISA assay kit, which reflected the transactivating function of two proteins on SV40 early promoter. The findings indicated that the expression of two viral proteins were successfully detected in soluble protein cell extracts of transiently transfected HepG2 cells. HCV NS3 protein transactivated the CAT enzyme expressed at a value 3 5 fold higher than the control, while HBX protein transactivated at a value 4 4 times. It arrived at 8 5 times when transfected with two plamids simultaneously. The activating effect was increased in relation to the amount of plasmids. It was suggested that the two kinds of viral proteins had a transactivating effect on SV40 early promoter, and they acted synergistically. These results might contribute to explaining the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection

Hepatitis C virus (HCV) non structure 3 (NS3) gene was amplified from plasmid pBRTM3011 by employing polymerase chain reaction (PCR), and the amplified product was cloned into pcDNA3 1(-) vector. Then the recombinant plasmid pcDNA3 1(-) NS3 was transfected into HepG2 cells and was cotransfected into HepG2 cells with reporter plasmid pCAT3 promoter, respectively. HCV NS3 protein expressed in HepG2 cells was detected by reverse transcription PCR (RT PCR) and Western blotting method. The activity of CAT was detected by a ELISA kit, which reflected the transactivating function of HCV NS3 protein. The results showed that HepG2 cells transfected with pcDNA3 1(-) NS3 could express HCV NS3 protein. The expression of CAT in HepG2 cells transfected with the pcDNA3 1(-) NS3 was 4 6 fold higher than that of control plasmid. It was suggested that the recombinant plasmid pcDNA3 1(-) NS3 could be expressed in mammalian cell line, and had transactivating effect on SV40 early promoter

Objective To explore the influence of XTP4 on genomic expression profile of hepatocyte through screening and cloning of genes trans-regulated by XTP4-expressing plasmid. Methods The expressive vector pcDNA3.1(-)-XTP4 was constructed by routine molecular biological methods, and suppression subtractive hybridization (SSH) method was employed to detect the mRNA differentially expressed by the HepG2 cells transfected with pcDNA3.1(-)-XTP4 and pcDNA3.1(-), respectively, using lipofectamine. The twice enriched PCR products were subcloned into T/A vectors to set up the subtractive library. Amplification of the library was carried out in E. coli strain JM109. The screened cDNA were sequenced and analyzed in GenBank with Blast search after PCR. Results The amplified subtractive library containd 21 positive clones. Colony PCR analysis showed that there were 16 clones containing 200-1000bp inserts. Sequence analysis was performed and 9 kinds of encoding sequences were achieved. These genes trans-regulated by XTP4 protein involved in hepatic fibrogenesis, tumorgenesis, mitochondrial function, and cell growth regulation. Conclusions The findings obtained by SSH provide significant data for a preliminary understanding of the biological function of a new identified gene-XTP4. These results will pave the way for the study of the molecular mechanism of the transactivating effects of HBxAg and the development of new therapy for chronic hepatitis B.

Objective To construct a subtractive cDNA library of genes differentially expressed in human lymphoma cell line Jurkat cells treated with glycyrrhizin (GL), and to clone genes associated with its immunological regulation, and to further elucidate the molecular immune mechanism of GL. Methods The mRNA was isolated from Jurkat cells treated with either GL or 0.9 percent sodium chloride as a control, then cDNA was synthesized. Suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The cDNA was sequenced and analyzed in GenBank with Blast search after the amplification of the subtractive library by PCR. Results The amplified library contained 28 positive clones. Colony PCR analysis showed that there were 22 clones containing 200-1 000 bp inserts. Sequence analysis was performed, and the full length sequences were obtained with bioinformatics method. Altogether 11 kinds of encoding sequences were achieved, including interleukin-12, interleukin-18, and thymosin ?1, etc. Conclusions A subtractive cDNA library of genes differentially expressed in Jurkat cells treated with GL using SSH technique was constructed successfully, and it might give some new clues for the study of the immune regulation mechanism of GL.