[Effect of OA kneepad on apoptosis genes Bcl-2 and p53 expression in articular cartilage cells of experimental knee osteoarthritis].

OBJECTIVE: To study the effects of kneepad on expression of Bcl-2 and p53 mRNA of chondrocyte in white rabbits with knee osteoarthritis, so as to explore and treatment mechanism of OA kneepad on apoptosis of chondrocytes of rabbits with knee osteoarthritis in molecular degree. METHODS: Forty-four Japanese healthy 6-month-old rabbits (equal male and female,the weight ranging from 2 to 2.2 kg) were used to establish knee osteoarthritis models by modified Hulth method. The rabbits were randomly divided into 6 groups: normal group, model group, control group (microwave), experimental group 1 (electricity), experimental group 2 (thermal), experimental group 3 (kneepad). Ten rabbits in the normal group were breed with conventional method; 9 rabbits in the model group were breed with conventional method after model made; 9 rabbits in the control group were treated with microwave for 30 minutes, one time daily; 9 rabbits in the experimental group 1 were treated with electricity (density wave) for 30 minutes,one time daily;8 rabbits in the experimental group 2 were treated with hot (hot soft membrane) for 30 minutes, one time daily; 9 rabbits in the experiment group 3 were treated with electrothermal (OA knee pad) for 30 minutes, one time daily. All the rabbits were treated for 16 weeks and then sacrificed. The expressions of Bcl-2 and p53 mRNA of chondrocytes in knee joint were detected by using fluorescence quantitative RT-PCR method. RESULTS: At the 16 hthek,th e OD260/OD280 value range of total RNA extracted from rabbit articular cartilage tissue in each group were all at 1.80 to 2.00,wh ich indicates high RNA purity. The p53 relative mRNA in articular cartilage cells of model group,th e control group,th e experimental group 1 ,r oup 2,gr oup 3 were overexpressed,an d Belc2 mRNA expression levels of articular cartilage cells were low expression,an d compared with the normal group there were significant differences (P < 0.01). Belc2, p53 mRNA expression in articular cartilage cells,th ere were significant differences (P < 0.01) between the control group, experimental group 1, group 2, group 3 and model group. The results between the control group, experimental group 1 ,group 2 and group 3 had significant differences (P < 0.01). CONCLUSION: OA-kneepad can up-regulate the mRNA expression of Bcl-2 as well as down-regulate the mRNA expression of p53, thereby to inhibit the apoptosis of cartilage cells and delay the degeneration of articular cartilage changes.

Increased expression of hLRH-1 in human gastric cancer and its implication in tumorigenesis.

Altered signaling pathways or deregulated transcription factors represent an important category of molecular events leading to aberrant gene regulation in gastric cancer, among which the role of WNT/beta-catenin pathway remains unclear. LRH-1 is a critical transcription factor in controlling cell proliferation via crosstalk with the beta-catenin signaling pathway. In order to gain a knowledge of the expression of hLRH-1v1 and hLRH-1 in gastric cancer, a Q-PCR analysis was carried out. Our results showed that in about 50 and 47.6% of 42 tested patients with gastric cancer, the mRNA expression of hLRH-1v1 and hLRH-1 was significantly upregulated, as compared with self-paired normal control, respectively. Besides, overexpression of hLRH-1 was shown to promote the proliferation of gastric adenocarcinoma cell SGC-7901 via induction of cyclin E1. Taken together, our present study demonstrated for the first time the increased expression of hLRH-1v1 and hLRH-1 in human gastric cancer, an alteration which may implicate in tumorigenesis.

Expression of CYP2E1 in human nasopharynx and its metabolic effect in vitro.

It was evident that nitrosamines can act directly on target tissue and result in carcinogenesis. As has been shown, the carcinogenic activity of nitrosamines relied on its bioactivation by Cytochrome P450 2E1 (CYP2E1). In this study, we investigated the expression of CYP2E1 in Nasopharyngeal carcinoma (NPC) cells, embryonic nasopharyngeal epithelial tissue (ENET) specimens, and NPC biopsies by RT-PCR analysis. CYP2E1 was expressed in all NPC cell lines (6/6, including 7429) and ENET (6/6), and 80% of NPC biopsie (8/10). The fact that Human nasopharynx expresses CYP2E1 suggests that CYP2E1 may play an important role in the course of NPC by indirect carcinogens nitrosamines. To further evaluate the function of CYP2E1, the CYP2E1 was stably expressed in the cell line NIH 3T3/rtTA under a tetracycline-controlled transactivator. The expression of CYP2E1 was tightly regulated in a dose-dependent manner by Doxycycline (Dox) When the catalytic activity of CYP2E1 was assayed, the result showed that the generation of 6-hydroxychlorzoxazone (6-OH-CZ) from chlorzoxazone (CZ) was dose- and time-dependent on Dox addition to the medium. In the presence of 1 microg/ml Dox, the CZ 6-hydroxylase activity of the cell line was found to be 0.986 +/- 0.034 nmol/10(6) cells/h. The metabolic activation of Tet/3T3/2E1-6 cells was also assayed by N,N'-dinitrosopiperazine (DNP) cytotoxicity, and the viability of Tet/3T3/2E1-6 cells treated with Dox was lower than that of untreated cells with a significant difference between them in 80 and 160 microg/ml DNP (P ( 0.05, t test. This cell line will be useful not only to assess the metabolic characteristics of CYP2E1, but also will be useful to investigate the role of CYP2E1 in metabolic activation of carcinogenic nitrosamines in vitro.

Microarray analysis of gene-expression profile in hepatocellular carcinoma cell, BEL-7402, with stable suppression of hLRH-1 via a DNA vector-based RNA interference.

To establish a cell line with a permanent suppression of hLRH-1 in this study, a stable RNAi vector (pSineohLRH-1) targeting hLRH-1 was constructed and introduced into hepatocellular carcinoma cell, BEL-7402. By semiquantitative RT-PCR analysis, the expression of hLRH-1 in BEL-7402 cells carrying pSineohLRH-1 was shown to be significantly suppressed by up to approximately 60%. In addition, microarray analysis was carried out to assess the extent of altered gene expression in BEL-7402 cells with stable knockdown of hLRH-1. Direct comparison of gene-expression profiles of more than 18,000 genes showed that 405 of the expressed genes in hLRH-1-knockdown cells differed dramatically in expression levels from those in controls, which suggested the even extensive biological functions of hLRH-1. Interestingly, among those differentially expressed genes, some are cancer-associated such as Gadd45beta and PTEN, and their expressions were further validated. Although the identification of the exact relationship between these genes and hLRH-1 awaits intensive investigation, the findings of this study provide new insights into the mechanism by which hLRH-1 is involved in tumorigenesis.

Experimental study on the suppression of human nuclear receptor hLRH-1 via a vector-based RNA interference.

To explore the inhibitions of human nuclear receptor hLRH-1 via RNA interference, siRNAs expressing vectors pShLRH-1.1 and pShLRH-1.2, and targeting hLRH-1 were designed and constructed. The recombinants were introduced into hepatocellular carcinoma cells, BEL-7402, mediated by lipofectamin. RT-PCR was carried out to examine the inhibition ratio of hLRH-1 expression. The same method was also applied to analyze the expression of farnesyl pyrophosphate synthetase (FPPS) gene. Our results demonstrated that after transient transfection, both pShLRH-1.1 and pShLRH-1.2 could trigger the efficient inhibition of hLRH-1 in cultured cells, BEL-7402. The inhibition ratios were up to 80%. By comparing with non-transfection and vector-transfection control, the expression of FPPS in cells with inhibition of hLRH-1 was up-regulated significantly. Thus, the inhibition of expression of hLRH-1 in cultured cells was achieved via RNA interference in this study. Our results also suggested that hLRH-1 acts as a negative regulator in FPPS expression.

Generation of transgenic mice with liver-specific expression of human nuclear receptor nr5a2.

Human nuclear receptor hb1 f(nr5a2) was cloned and characterized as a novel member of the Ftz-F1 (nr5a) nuclear receptor subfamily,whose its biological function remained largely unidentified. The aim of this study was to establish transgenic mouse model that specifically expressed hB1F in the liver to faciliate the functional study of hB1F. hb1f cDNA was placed downstream of mouse albumin gene enhancer/promoter to construct a liver-specific hb1f expression vector. Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. Four offspring were identified as carrying the transgenes by PCR,from which one was also verified by Southern blotting. RT-PCR and Western blotting results showed that the transgene was expressed in the liver of the transgenic mice. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 mice were identified by PCR analysis. Genetic analysis of the transgenic mice demonstrated that the transgene had been integrated into the chromosome at a single site and could be stably transmitted.

[Progress in nuclear receptor research].

Nuclear receptors belong to a superfamily of ligand-activated transcription factors, which are involved in regulating gene expression in development, cell differentiation, physiological and metabolic processes. In this review we summarize the studies of nuclear receptor and present the progresses in the researches on nuclear receptor and lipid physiology, nuclear receptor and tumor, and nuclear receptor and co-regulators.

Gene expression profile in liver of hB1F transgenic mice.

AIM: To analyze the tissue morphologic phenotype and liver gene expression profile of hB1F transgenic mice. METHODS: Transgene expression was analyzed with RT-PCR and Western blotting. For one of the transgenic mouse lines, tissue expression pattern of the transgene was also examined with immunochemical methods. Pathological analysis was used to examine the tissue morphologic phenotype of established transgenic mice. The liver gene expression profile of transgenic mice was analyzed with microchip, and some of the differentially expressed genes were verified with RT-PCR. RESULTS: The expressions of hB1F were shown in livers from 6 of 7 transgenic mouse lines. The overexpression of hB1F transgene did not cause pathological changes. Expressions of three genes were up-regulated, while down-regulation was observed for 25 genes. CONCLUSION: The overexpression of hB1F transgene may cause changes of gene expression profiles in the liver of transgenic mice.

Establishment of transgenic mice carrying gene encoding human zinc finger protein 191.

AIM: Human zinc finger protein 191 (ZNF191) was cloned and characterized as a Kruppel-like transcription factor, which might be relevant to many diseases such as liver cancer, neuropsychiatric and cardiovascular diseases. Although progress has been made recently, the biological function of ZNF191 remains largely unidentified. The aim of this study was to establish a ZNF 191 transgenic mouse model, which would promote the functional study of ZNF191. METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. The offsprings were identified by PCR and Southern blot analysis. ZNF 191 gene expression was analyzed by RT-PCR. Transgenic founder mice were used to establish transgenic mouse lineages. The first generation (F1) and the second generation (F2) mice were identified by PCR analysis. Ten-week transgenic mice were used for pathological examination. RESULTS: Four mice were identified as carrying copies of ZNF191 gene. The results of RT-PCR showed that ZNF 191 gene was expressed in the liver, testis and brain in one of the transgenic mouse lineages. Genetic analysis of transgenic mice demonstrated that ZNF 191 gene was integrated into the chromosome at a single site and could be transmitted stably. Pathological analysis showed that the expression of ZNF 191 did not cause obvious pathological changes in multiple tissues of transgenic mice. CONCLUSION: ZNF 191 transgenic mouse model would facilitate the investigation of biological functions of ZNF191 in vivo.

Establishment of transgenic mice carrying the gene of human nuclear receptor NR5A2 (hB1F).

AIM: Human hepatitis B virus enhancer II B1 binding factor (hB1F) was cloned and characterized as a novel member of the Ftz-F1 (NR5A) nuclear receptor subfamily. Although progresses have recently been made, its biological function remains largely unidentified. The aim of this study was to establish an hB1F transgenic mouse model to promote the functional study of hB1F. METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. The offsprings were identified by PCR and Southern blot analysis. Transgene expression was analyzed with RT-PCR and Western blot analysis. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 and F2 mice were identified by PCR analysis. RESULTS: Seven mice were identified as carrying copies of transgene. RT-PCR and Western blotting results showed that the transgene was expressed in heart, liver, lung, kidney and stomach in one of the transgenic mouse lineages. Genetic analysis of the transgenic mice demonstrated that the transgene was integrated into the chromosome at a single site, and was transmitted stably. CONCLUSION: In this study we established an hB1F transgenic mouse model, which will facilitate the investigation of the biological function of hB1F in vivo.

[Cloning, genomic organization and promoter activity of the mouse zinc finger protein gene ZF-12].

The human zinc finger protein ZNF191 is a krüppel-like transcription factor, which may be relevant to many diseases such as neuropsychiatric, cardiovascular and liver caner diseases. To elucidate the function of ZNF191 by gene targeting, it is necessary to clone and characterize of the homologous gene in model organisms (mice). The mouse homologous gene (ZF-12) was cloned and sequenced for the first time, the GenBank accession number is AY052495. It contains four exons and three introns; all intronic splice sites exhibited consensus GT/AG sequences. The single nucleotide polymorphisms (SNPs) in exon 2 and the alternative length of 3'-untranslated region (3'-UTR) have been found. The linkage of the ZF-12 gene and the zinc finger protein gene Zfp-35 has been found, so the ZF-12 gene can be localized to B3 to C or beside of chromosome 18. We assessed approximately 1.2 kb of 5'-flanking region of the ZF-12 gene for basal promoter activity. A series of deletion mutants of 5'-flanking region linked to the luciferase gene was constructed. Basal level expression of these constructs was tested in COS-7 cells, NIH3T3 cells and HeLa cells. By measuring luciferase activity, which was transiently expressed in the transfected cells, we found that regulatory elements sufficient for basal expression lie between -762 and +70 bp relative to the transcription start site and that a negative regulatory region lie between -824 and -762 bp. This research provides a basis for further study on ZF-12 by gene targeting.

[Expression of human cytochrome P450 2E1 gene in embryonic nasopharynx, nasopharyngeal cancer cell lines and tissue].

BACKGROUND & OBJECTIVE: Research results in the past suggested that nitrosamines(chemicals) can induce nasopharyngeal carcinogenesis in vivo and in vitro. As the indirect carcinogens, their activation are mainly dependent on cytochrome P450 2E1. The study here was to investigate the roles of human cytochrome P450 2E1 (hCYP 2E1) gene in chemical cancinogen-induced nasopharyngeal carcinogenesis, and to provide new evidence about etiology and pathogenesis of nasopharyngeal carcinoma(NPC). METHODS: RT-PCR were used to detect the expression of CYP2E1 in 8 embryonic nasopharyngeal epithelial tissues(ENET), 10 NPC biopsys, 5 NPC cell lines(CNE-1, CNE-2, HNE-1, HNE-2, and HNE-3), 1 transformed nasopharyngeal cell line(7429) and 3 human embryonic nasopharyngeal epithelial(HENE) cell lines treated with 200, 250, and 300 micrograms/ml of chemical carcinogen N, N'-dinitrosopiperazine(DNP) in vitro for 24 h, respectively. RESULTS: (1) All of ENET(8/8), NPC cell lines(6/6, including 7429 cell line) and 80% of NPC biopsys(8/10) showed expression of hCYP2E1; (2) The hCYP 2E1 expression in HENE cell lines was elevated after treatment with different concentration of DNP. CONCLUSION: Human nasopharynx exist inducible CYP2E1 expression, suggesting that hCYP2E1 may play an important role in the course of nasopharyngeal carcinogenesis caused by indirect carcinogens nitrosamines(such as DNP).

[Cloning and identification of a mouse zinc finger protein gene ZF-12-related pseudogene].

The mouse zinc finger protein ZF-12 gene is homologous to human gene and encodes a protein of 368 amino acids, which contains four tandem C2H2-type zinc finger motifs in the N-terminal and one SCAN domain in the C-terminal. Some recent studies suggest that ZNF191 might be a hepatocarcinogenesis-associated gene. We screened a mouse lambda genomic library with a human ZNF191 cDNA probe and isolated a ZF-12-like gene, named ZF12p (GenBank AY040222). This intronless gene closely resembles ZF-12 but displays several mutations, suggesting that ZF12p represents a ZF-12-related pseudogene. RT-PCR analysis on total RNA from mouse tissue and bioinformatis analysis on promoter region of ZF12p gene, suggest the transcripts of ZF12p may be not synthesized. BLAST on the data of the human genome in the GenBank with ZNF191 cDNA and Southern blotting show there is no any psedogene related to ZNF191 gene in the human genome. The high similarity of ZF12p to ZF-12 might be of considerable importance for mutation and evolution analysis of ZF-12.