Amaxa Nucleofector(TM) nuclear transfection apparatus transfers L1210 cell line / 中国实验血液学杂志

Mouse L1210 leukemia cell line is widely used as a model in the study of tumorigenesis, as well as the efficacy of chemotherapeutic drugs; however, like other suspension cell lines, the mouse L1210 cell line has lowest transfection efficiency, that many barriers exist to study about the structure, function, as well as metabolism in leukemia cells. This study was aimed to obtain higher transfection efficiency of L1210 cell line to facilitate scientific research. The transfection efficiencies of nucleofector and liposome in L1210 leukemia cells were detected by converted fluorescence microscopy and flow cytometry using EGFP (enhance green fluorescent protein); cell viability was observed by trypan blue exclusion test. The results showed that the transfection efficiency of nucleofector primarily through reporter gene pEGFP by Amaxa Nucleofector(TM) nuclear transfer apparatus was significantly higher than lipofectamine 2000 transfection, furthermore, in the same cell density (2 × 10(6)/ml) and plasmid content (10 µg), the transfection efficiency of nuclear transfer apparatus default mode A-20 was higher than that of other modes (S-18, T-20). Its survival rate was up to 50.5% after 24 hours. Cell viability of liposome transfection reached to 88% after 24 hours, but the transfection efficiency was lower (< 1%). It is concluded that the nuclear transfer apparatus A-20 transfected L1210 can reach higher transfection efficiency up to 61.6%, which is significantly higher than that of lipofectamine transfection. The survival rate is up to 50.5% well meeting the needs of scientific research. Higher transfection efficiency is helpful for in-depth research about the morphology, functions and pathogenesis in leukemia model L1210, and provides more searching space for the treatment of leukemia diseases.

Effect of silencing connective tissue growth factor on rat liver fibrosis and the accumulation of extracellular matrix / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the anti-fibrogenesis property of intraportal vein injection of small interfering RNA targeting connective tissue growth factor (CTGF) in a rat model of liver fibrosis and its effect on the accumulation of extracellular matrix (ECM).</p><p><b>METHODS</b>Thirty male rats were randomly divided into five groups. Some rats received CCl4 subcutaneously every three days for 6 consecutive weeks, and in the meantime they also received either siRNA targeting CTGF (preventive group), saline (model group) or siRNA (siRNA control group) by intraportal vein injections. Other rats received CCl4 by subcutaneous injection for 2 weeks, followed by CCl4 and CTGF siRNA intraportal vein injection for 4 more weeks (as treatment group). The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot respectively. Hepatic histology was evaluated by HE and Sirius red stained sections. The collagen staining areas were measured quantitatively using a computer-aided manipulator with slight modifications. Serum procollagen type III and hyaluronic acid were determined by radioimmunoassay.</p><p><b>RESULTS</b>Six weeks after CCl4 injection, prominent upregulation of gene expressions of CTGF, type I and III collagen, and laminin in saline or siRNA-treated rat livers were observed. The expressions of CTGF at mRNA and protein level and type I and III collagen at mRNA level were markedly reduced in rats with CTGF siRNA treated for four or six weeks. Expressions of CTGF at mRNA and protein levels decreased by 76%+/-8%, 80%+/-3% (F = 68.630) and 95%+/-2%, 93%+/-3% (F = 21.234, P < 0.01); type I and III collagen and laminin at mRNA levels decreased by 74%+/-8%, 78%+/-8%, 31%+/-7% and 57%+/-6%, 59%+/-10%, 43%+/-9% (F = 24.219, 16.315, 9.716, P < 0.01) compared with rats in the model group at 72 h. The CTGF siRNA treatment markedly reduced serum levels of procollagen type III and hyaluronic acid and the degrees of liver fibrosis.</p><p><b>CONCLUSION</b>Intraportal vein siRNA injection targeting CTGF could significantly inhibit CTGF gene expression in rats, thereby attenuating liver fibrosis by reducing ECM accumulation.</p>

Preparation and identification of hammerhead ribozyme in vitro against caspase-12 mRNA fragments / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To design and synthesize ribozymes targeting 138 and 218 sites of the mRNA nucleotide of mouse caspase-12, a key intermedium of ER stress mediated apoptosis, and to identify their activities through in vitro transcription and cleavage.</p><p><b>METHODS</b>The mouse caspase-12 gene fragment was obtained by RT-PCR and cloned into the PGEM-T vector under the control of T7 RNA polymerase promoter. The transcription product of the target was labeled with a-32P UTP, while ribozymes were not labeled. Ribozyme and target RNA were incubated for 90 min at 37 degree C in a reaction buffer to perform the cleavage reaction.</p><p><b>RESULTS</b>It was found that under a condition of 37 degree C, pH 7.5 and with Mg2+ in a concentration of 10 mmol/L, Rz138 and Rz218 both cleaved targets at predicted sites, and the cleavage efficiency of Rz138 was 100%.</p><p><b>CONCLUSION</b>Rz138 and Rz218 prepared in vitro possess the perfect specific catalytic cleavage activity. Rz138 has excellent cleavage efficiency. It may be a promising tool to prevent ER stress induced apoptosis through catalytic cleavage of caspase-12 mRNA in vivo. It also can be used to verify whether caspase-12 is necessary in ER stress induced apoptosis.</p>

Effects of ribozyme targeting platelet-derived growth factor receptor beta subunit gene on the proliferation and apoptosis of hepatic stellate cells in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor beta subunit (PDGFR-beta) is the predominant signal transduction pathway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-beta mRNA in HSC and the effect on biological characteristics of HSC.</p><p><b>METHODS</b>Expression vector of anti-PDGFR-beta ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-beta, alpha-smooth muscle actin, and typeI and type III collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.</p><p><b>RESULTS</b>The expression of PDGFR-beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49% - 57% (P < 0.05 - 0.01). The proliferation and alpha-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased (P < 0.05 - 0.01), and the type I and type III collagen synthesis were also reduced (P < 0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC (P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy.</p><p><b>CONCLUSIONS</b>The anti-PDGFR-beta ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-beta expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-beta expression.</p>

Inhibition of mutant-type p53 by a chimeric U6 maxizyme in hepatocellular carcinoma cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines.</p><p><b>METHODS</b>Maxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot.</p><p><b>RESULTS</b>In vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis.</p><p><b>CONCLUSION</b>Our findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.</p>

Inhibition of mouse hepatocyte apoptosis by anti-caspase-12 small interfering RNA / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the inhibition of primary mouse hepatocyte apoptosis by small interfering RNA (siRNAs) against caspase-12.</p><p><b>METHODS</b>The Balb/c mouse primary hepatocytes were isolated in situ with two-step liver perfusion with 0.5 g/L collagenase type IV, and apoptosis were induced with 4 micromol/L thapsigargin (TG). The three kingds of siRNAs targeting different gene sites (130, 214, 521) were synthetized chemically. The single-stranded RNAs were annealed to produce double-stranded siRNAs, then the mouse primary hepatocytes were transfected by oligofectamine package. The inhibition of caspase-12 was analyzed with RT-PCR and Western-blot. The viable hepatocytes following the induction of apoptosis were evaluated with MTT.</p><p><b>RESULTS</b>All the three kinds of siRNAs could obviously inhibit normal mouse hepatocyte caspase-12 mRNA. The siRNA (214) were more effective than the other two when the concentration was 100 nmol/L. The caspase-12 mRNA expression was inhibited by 52.08%, while that of siRNA (521) was 30.73% (t=4.30, P <0.05). However when the concentration was 200 nmol/L, the inhibitions were similar (88.07%, 86.22% and 89.41% respectively). siRNA (214) could downregulate the expression of apoptotic hepatocytes procaspase-12 by 51.43% ( t=4.30, P <0.01). Contrasted with apoptotic hepatocytes, the cell activity, which was analyzed with MTT, increased by 48.76% (t=2.23, P <0.01).</p><p><b>CONCLUSION</b>siRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.</p>

The expression of platelet-derived growth factor (PDGF) receptor-beta and its correlation with extracellular matrix in hepatic tissue in hepatic fibrosis rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expression of PDGF receptor-beta and its correlation with extracellular matrix in hepatic tissue during hepatic fibrosis.</p><p><b>METHODS</b>The model of hepatic fibrosis in rats was induced by carbon tetrachloride. PDGF receptor-beta subunit, collagen I, collagen III and a-SMA in hepatic tissues of these rats were examined using immunohistochemistry. The correlation between PDGF receptor-beta subunit and collagen I, III was analyzed using SAS software after the results of immunohistochemistry were semi-quantified.</p><p><b>RESULTS</b>PDGF receptor-beta subunit and a-SMA were not detected in normal controls. Collagen I and III were distributed in the portal tracts and beneath the endothelia of the central veins and of the Disse spaces. Two weeks after CCl4 injection, the PDGF receptor-beta and a-SMA were detected, and the expression of collagen I and III increased. At the end of 4 and 6 weeks, the above four proteins were further increased. Two weeks after CCl4 injection, PDGF receptor-beta had no apparent correlation with collagen I and III. However, PDGF receptor-beta had a significant correlation with collagen I and III 2 weeks later, and the correlation coefficient was 0.74 and 0.60 respectively at 4 weeks, and 0.83 and 0.67 respectively at 6 weeks. PDGF receptor-beta had a significant correlation with a-SMA during the whole process of hepatic fibrosis and the correlation coefficient was 0.62, 0.69 and 0.81, respectively at the time of 2, 4 and 6 weeks after CCl4 injection.</p><p><b>CONCLUSION</b>The PDGF receptor-beta was overexpressed during the process of hepatic fibrosis development, and it significantly correlated with collagen I and collagen III.</p>

Clone, expression and cleavage activity of anti-caspase-7 hammerhead ribozyme in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To design hammerhead ribozymes against mouse caspase-7 and to study their expression and cleavage activity in vitro.</p><p><b>METHODS</b>The secondary structures of ribozyme and caspase-7 genes were analyzed and simulated by computer. Ribozymes DNA sequences were synthesized by automatic synthetic apparatus. Caspase-7 DNA sequence was acquired by reverse transcription PCR. Ribozymes and caspase-7 DNA sequences were separately cloned into pBSKneo U6 and pGEM-T vectors. Ribozymes and caspase-7 mRNA were obtained by transcription in vitro, and ribozymes cleavage activity was identified by cleavage experiment in vitro.</p><p><b>RESULTS</b>Two ribozymes named Rz333 and Rz394 targeting 333 and 394 sites in caspase-7 mRNA were designed by computer software, and their DNA sequences were synthesized. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed by in vitro transcription. In vitro cleavage experiments showed that Rz333 cleaved caspase-7 mRNA and produced 243nt and 744nt segments. The cleavage efficiency is 67.98%, while Rz394 cannot cleave caspase-7 mRNA.</p><p><b>CONCLUSIONS</b>Rz333 can site-specifically cleave caspase-7 mRNA.</p>

Effect of small interfering RNA targeting connective tissue growth factor on the synthesis and secretion of extracellular matrix in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of chemically synthetic small interfering RNA (siRNA) targeting connective tissue growth factor (CTGF) on the synthesis and secretion of extracellular matrix (ECM) in hepatic stellate cells (HSC).</p><p><b>METHODS</b>Chemically synthetic siRNA targeting CTGF was transfected into HSC T6 (an active HSC line in rats) by oligofectamine package, and untreated HSC T6 were used as control. Total RNA and protein of the cells, after their incubation with siRNA for 24, 48 and 72 hours, were extracted, and the supernatants were collected. The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot. Contents of hyaluronic acid and type III pro-collagen in the supernatants were determined by radioimmunoassay.</p><p><b>RESULTS</b>The expression of CTGF at mRNA and protein level and type I and III collagen at mRNA levels were markedly down-regulated in siRNA-transfected HSCs. The contents of hyaluronic acid and type III pro-collagen in the supernatants decreased by 46%+/-7%, 52%+/-7%, 53%+/-7% and 29%+/-18%, 29%+/-7%, 27%+/-5%, compared with those of the blank control at 24, 48 and 72 hours.</p><p><b>CONCLUSIONS</b>Chemically synthetic anti-CTGF siRNA can significantly inhibit CTGF gene expression in HSC, and markedly reduce the synthesis and secretion of ECM including type I and III collagen and hyaluronic acid. The siRNA-directed suppression of CTGF gene in HSC was maintained for 72 hours. This suggests that chemically synthetic siRNA may be a potential in preventing and treating liver fibrosis and may have a promising future for development</p>

Effect of ribozyme against platelet-derived growth factor receptor beta subunit mRNA on the biological characters of hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the cleavage activity of hammerhead ribozyme targeting at platelet-derived growth factor receptor beta subunit (PDGFR- beta) mRNA in hepatic stellate cells (HSCs) and its effect on the biological characters of HSCs.</p><p><b>METHODS</b>Expression vector of anti-PDGFR- beta ribozyme was constructed and transfected into rat-derived HSC-T6 cells with lipofectin. The positive cell clones were gained by G418 selection. The expression of PDGFR- beta, alpha-smooth muscle actin (alpha-SMA), and type I and type III collagen was detected by means of northern blot, Western blot and immunocytochemical staining respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was demonstrated with flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.</p><p><b>RESULTS</b>The expression of PDGFR- beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSCs only 43% to 51% of that in control cells (t > or = 3.957, P < 0.05), and alpha-SMA expression level, type I and type III collagen synthesis ability were also reduced (t > or = 6.790, P < 0.01). The proliferation of ribozyme-transfected HSCs was significantly decreased (t > or = 3.858, P < 0.05), and the proliferation response to PDGF BB was markedly inhibited. However the apoptotic rate was significantly increased in ribozyme-transfected HSCs (chi2 > or = 14.157, P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy.</p><p><b>CONCLUSIONS</b>The anti-PDGFR- beta ribozyme can be expressed stably in HSCs, cleave the target RNA effectively, inhibit HSCs proliferation and collagen synthesis, and induce HSC apoptosis. The results suggest that inhibiting PDGFR- beta expression in HSCs may be a new therapy for liver fibrosis.</p>

Inhibition of mutant p53 in hepatocellular carcinoma cells by hammerhead ribozyme / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the inhibition of mutant type p53 in hepatocellular carcinoma by hammerhead ribozyme in both cell-free system and MHCC97 cells.</p><p><b>METHODS</b>Hammerhead ribozyme genes (RZ) and control ribozyme (asRZ) directed against mutant p53 (249 codons, AGG --> AGT) were designed by computer. The in vitro transcription plasmid and eukaryotic expression plasmid were constructed into the vector pBSKU6 and pEGFPC1. Human mutant and wild type p53 gene fragment were cloned into the pGEM-T vector under T7 promoter control. In vitro cleavage reaction was carried out by mixing the RZ and target mRNAs which were labeled with [alpha-32P] dUTP. RZ was introduced into MHCC97 cells by LipofectAMINEAM2000 and mtp53 expression was analyzed by RT-PCR.</p><p><b>RESULTS</b>In cell-free systems, RZ showed a specific cleavage activity against mtp53 with cleavage efficiency of 42%, while the wild type p53 was not cleaved. The mRNA level of mtp53 in MHCC97 cells after transfection was reduced by RT-PCR analysis.</p><p><b>CONCLUSION</b>These findings suggest that the hammerhead ribozyme against the mtp53 is a new promosing gene therapeutic agent against hepatocellular carcinoma.</p>

Construction and identification of a specific small interfering RNA expression vector of Caspase-12 in mouse hepatoma cell line / 中华传染病杂志

Objective To construct a specific small interfering double-stranded RNA(siRNA) expression vector of Caspase-12 and to evaluate inhibitory effect of this siRNA on caspase-12 mRNA activity.Methods Three groups of siRNA targeting different gene sites of caspase-12 were designed and synthesized chemically.Mouse hepatoma cell line,Hepa1-6,was transfected with the siRNA by 24 h.Reverse transcription-polymerase chain reaction(RT-PCR)was performed to analyze the inhibi- tion of caspase-12.Then the most effective siRNA was selected and the two template sequences for the siRNA were inserted into pRNAT-H1.1Neo expression vector.The recombinant plasmid, referred to as pRNAT-casp12,was verified by PCR analysis and sequencing.The expression of caspase-12 at mRNA and protein level,after transfection with pRNAT-casp12 by 48 h and 72 h respectively,were analyzed by using real-time PCR and Western blotting.Results The chemically synthesized siRNA*1 and siRNA*3 could inhibit mouse hepatoma cell caspase-12 mRNA by 59.9% and 39.6%(P