Construction and identification of mouse Krüppel-like factor 4 recombinant lentiviral expression vector and its effect on proliferation of RAW264.7 cells / 天津医药

Objective To construct and identify a lentiviral vector carrying mouse Krüppel-like factor 4 (KLF4) gene, and establish RAW264.7 cell line of peritoneal macrophages that over-expressed KLF4. Methods KLF4 gene was cloned using the measure of polymerase chain reaction (PCR). Then the recombinant transfer vector pLVX-KLF4 (pLVX-KLF4-mCMV-ZsGreen-PGK-Puro) was constructed. The pLVX-KLF4 was confirmed through PCR, restriction enzyme digestion and sequencing. The correct recombinant transfer vector together with its two helper virus vectors (psPAX2 and pMD2.G) were cotransfected into the 293T cells by Lipofectamine? 3000. The supernatant of 293T was harvested to infect RAW264.7 cells. Flow cytometry (FCM) was used to test the viral titer of the expression level of green fluorescent protein. The expression of KLF4 mRNA in RAW264.7 cells was measured by real-time PCR. Results The restriction enzyme digestion, PCR and sequencing confirmed that the transfer lentiviral vector pLVX-KLF4 was constructed successfully. KLF4 mRNA was over-expressed in Lenti-KLF4 transfected RAW264.7 cells than that of wild type RAW264.7 cells (P<0.05). In transfected RAW264.7 cells, KLF4 mRNA was over-expressed (P<0.05). The recombinant lentivirus of KLF4(Lenti-KLF4)titer was 2.05×108 TU/mL measured by FCM.The flow cytometry results showed that the S phase fraction was prolonged and G0/G1 was arrested in the over-expressed KLF4 of RAW264.7 cells. The EdU showed that the up-regulated expression of KLF4 gene stimulated the proliferation of RAW264.7 cells. Conclusion The recombinant lentiviral vector, which can effectively express KLF4 mRNA, has been successfully constructed. The up-regulated KLF4 gene may increase the proliferation of RAW264.7 cells.

The dynamic changes of collagen fiber in quartz-induced pulmonary fibrosis in mice with picrosirius red plus polarized microscopy in vivo / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the use value of picrosirius red staining plus polarized microscopy to observe the dynamic changes of collagen fiber in lung fibrosis in silicotic mice model.</p><p><b>METHODS</b>The experimental mice were divided into control and quartz groups. 0.2 g/kg weight of quartz was injected intratracheally in quartz group. Lung tissues were collected at the 1st, 3rd, 5th, 7th, 14th and 28th day after injection respectively. Lung tissue slides were stained with picrosirius red. With the aid of polarized microscope, image analysis software, the distribution and change of type I and type III collagen could be qualitatively and quantitatively analyzed. Lung tissue hydroxyproline was determined by chloramines T method.</p><p><b>RESULTS</b>In early stage the predominant increment was type III collagen, but in late stage type I was predominant. The contents of both type collagen tended to increase as postexposure time prolonged. The time course of the ratio of type I to type III showed increasing trend, and there was a statistical significance on day 28 (1.49 +/- 0.39 vs 0.59 +/- 0.24, P < 0.05). The total area of collagen was positively correlated with hydroxyproline concentration of lung tissue (r(2) = 0.928 5, P < 0.01).</p><p><b>CONCLUSION</b>Picrosirius red staining combined with polarized microscopy and digital image processing is a useful method to elucidate collagen accumulation, distribution and subtype ratio in silicosis.</p>

Expression of Smads in lung tissue of quartz-induced pulmonary fibrosis in mice / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To observe Smads protein expression in lung tissue of quartz exposed mice and to explore its association with pulmonary fibrosis in silicosis.</p><p><b>METHODS</b>The experimental mice were divided into control and quartz groups. 0.2 g/kg weight of quartz was injected intratracheally in quartz group. Samples were collected at the 1st, 3rd, 5th, 7th, 14th and 28th day after injection. Immunohistochemical methods with quantitative image analysis were used to assay the protein expression of transforming growth factor beta(1) (TGF-beta(1)), Smad 2/3, Smad 4, and Smad 7 protein levels. Protein expression level is presented by positive unit (PU).</p><p><b>RESULTS</b>Smad 2/3 protein expression increased from day 3, reaching its peak level in day 14 [(42.2 +/- 2.4) PU], and decreased gradually. The elevation of Smad 4 protein level began from day 5, and the highest degree came into day 14 [(40.0 +/- 1.8) PU], decreased thereafter. The expression of Smad 7 presented a decreasing tendency at the beginning and reaching the lowest level in day 14 [(33.5 +/- 3.3) PU]. It seemed to elevate in day 28, but was still lower than the controls. There were positive correlation between Smad 2/3, Smad 4 and TGF-beta(1) (r = 0.91, r = 0.71, respectively, P < 0.05) and also between Smad 2/3 and hydroxyproline contents of lung tissue (r = 0.85, P < 0.05) except Smad 7.</p><p><b>CONCLUSION</b>Smad protein may have certain association with pulmonary fibrosis in silicosis.</p>

RT-PCR method for detecting the expression of transforming growth factor-beta1 gene in lung tissues of silica-treated mice / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the gene expression of transforming growth factor-beta(1) (TGF-beta(1)) in lung tissues of silica-treated mice.</p><p><b>METHODS</b>The experimental mice were divided into control and silica group. 0.2 g/kg body weight of silica was injected intratracheally in silica group. Samples of lung tissue were collected 1, 3, 5, 7, 14 and 28 d after injection. RT-PCR method was used to analyze the gene expression of TGF-beta(1) in lung tissue of silica-treated mice.</p><p><b>RESULTS</b>The expression of TGF-beta(1) gene in lung tissue elevated from the 3rd day (1.20 +/- 0.15) and the peak value was on the 7th day (1.74 +/- 0.19). Then the expression decreased from the 14th to 28th day. But there was still higher than control until the 28th day.</p><p><b>CONCLUSION</b>TGF-beta(1) may play an important role in silica-induced pulmonary fibrosis.</p>

Immunohistochemical method for the detecting expression of transforming growth factor-beta1 in lung tissues of silica-treated mice / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the protein expression of transforming growth factor-beta(1) (TGF-beta(1)) in lung tissues of silica-treated mice.</p><p><b>METHODS</b>The experimental mice were divided into control and silica groups. 0.2 g/kg body weight of silica was injected intratracheally in mice of silica group. Samples of lung tissue were collected 1, 3, 5, 7, 14 and 28 d after injection. The immunohistochemical method was used to analyze the protein expression of TGF-beta(1).</p><p><b>RESULTS</b>In control mice, the expression of TGF-beta(1) in lung tissue was slightly positive while it was markedly increased in silica-treated mice. The expression was significantly elevated from the 7th day to 14th day. The expression in alveolar macrophages reached the peak on the 5th day [(93.4% +/- 2.8%) vs (42.2% +/- 12.0%), P < 0.01].</p><p><b>CONCLUSION</b>TGF-beta(1) may play an important role in early development of silicosis.</p>