The effects of gossypol on expressions of transforming growth factor-β1, fibronectin and 11β-hydroxysteroid dehydrogenase in nephridial tissue in rats with diabetes mellitus

Objective To observe the effects of gossypol on expressions of transforming growth factor-β1 (TGF-β1), fibronectin (FN) and 11β-hydroxysteroid dehydrogenase (11β-HSD) in nephridial tissue in rats with diabetes mellitus. Methods Thirty male Sprague-Dawley rats were randomly divided into three groups: normal control, type 2 diabetes and gossypol treatment group . After high-fat feeding for 4 weeks, the later two groups were injected with low dosage strepozotocin (30 mg/kg) intraperitoneally to induce type 2 diabetic rat model. The rats in gossypol treated group were given gossypol at the dosage of 15 mg/kg once per day for 4 weeks by gavage. And since the 5th week, the times of gavages had been changed into once per week at the same dosage and lasted to the 12th week . The levels of blood glucose, total cholesterol (TC), low density lipoprotein-cholesterol (LDL-c) were measured. Additionally, the morphological changes of the kidney were studied by light microscopy and transmission electron microscopy respectively. The mRNA expressions of TGF-β1, FN, 11β-HSD and 11β-HSD2 in nephridial tissue were assayed by semi-quantity RT-PCR. The protein expressions of TGF-β1 and FN in nephridial tissue were determined by immunohistochemistry. Results The blood levels of glucose, TC and LDL- c were increased significantly in type 2 diabetic group compared with normal control group(P<0. 01). The volume of glomerulus and the deposition of PAS positive substance in the glomerular interstitium were increased under light microscopy, and the glomerular basal membrane was thicker in type 2 diabetic group than those in normal control group under transmission electron microscopy. The mRNA and protein expressions of TGF-β1 and FN were increased(P<0. 01), and the mRNA expression of 11β-HSD2 was decreased(P<0. 05), while the mRNA expression of 11β-HSD1 was unchanged in type 2 diabetic group compared with normal control group. After the treatment of gossypol, the level of the blood glucose was significantly decreased(P< 0. 01), and the levels of TC, LDL-c showed a trend of decrease but had no statistical differences compared with type 2 diabetic group. The morphology of nephridial tissue was ameliorated in gossypol treatment group. The mRNA and protein expressions of TGF-β1 and FN were decreased(0. 16± 0. 02,0. 22±0. 05 ; 0. 24±0. 06,0. 33±0. 07, P< 0. 05), while the mRNA expressions of 11β-HSD1 and 11β-HSD2 were unchanged compared with type 2 diabetic group. Conclusions Gossypol can relieve the pathologic changes of nephridial tissue, inhibit the expressions of TGF-β1 and FN through decreasing blood glucose of rats with diabetes mellitus.     

改良小腿筋膜蒂皮瓣修复皮肤缺损的临床应用

目的 探讨改良的小腿筋膜蒂皮瓣修复皮肤缺损的临床应用.方法 对6例小腿皮肤缺损钢板或骨外露创面患者,缺损面积：1.8 cm×3.0 cm～3.2 cm×6.5 cm,分别以改良的小腿筋膜蒂转移皮瓣修复,经过精确测量,将皮瓣通过皮下隧道转移至受区覆盖皮肤缺损创面,供区直接缝合.结果 6例皮瓣全部成活,供区仅线性瘢痕.术后随访2个月～2年,平均7个月,小腿功能良好,皮瓣外观满意,皮肤质地好.结论 改良的小腿筋膜蒂皮瓣在修复皮肤缺损的临床应用中是一种简单、安全、理想的手术方式.     

大鼠肝细胞生长因子与肝再生增强因子融合基因真核表达质粒的构建及鉴定

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.     

大鼠肝细胞生长因子与肝再生增强因子融合基因真核表达质粒的构建及鉴定

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.     

大鼠肝细胞生长因子与肝再生增强因子融合基因真核表达质粒的构建及鉴定

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.     

大鼠肝细胞生长因子与肝再生增强因子融合基因真核表达质粒的构建及鉴定

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.     

The effects of gossypol on expressions of transforming growth factor-β1, fibronectin and 11β-hydroxysteroid dehydrogenase in nephridial tissue in rats with diabetes mellitus

Objective To observe the effects of gossypol on expressions of transforming growth factor-β1 (TGF-β1), fibronectin (FN) and 11β-hydroxysteroid dehydrogenase (11β-HSD) in nephridial tissue in rats with diabetes mellitus. Methods Thirty male Sprague-Dawley rats were randomly divided into three groups: normal control, type 2 diabetes and gossypol treatment group . After high-fat feeding for 4 weeks, the later two groups were injected with low dosage strepozotocin (30 mg/kg) intraperitoneally to induce type 2 diabetic rat model. The rats in gossypol treated group were given gossypol at the dosage of 15 mg/kg once per day for 4 weeks by gavage. And since the 5th week, the times of gavages had been changed into once per week at the same dosage and lasted to the 12th week . The levels of blood glucose, total cholesterol (TC), low density lipoprotein-cholesterol (LDL-c) were measured. Additionally, the morphological changes of the kidney were studied by light microscopy and transmission electron microscopy respectively. The mRNA expressions of TGF-β1, FN, 11β-HSD and 11β-HSD2 in nephridial tissue were assayed by semi-quantity RT-PCR. The protein expressions of TGF-β1 and FN in nephridial tissue were determined by immunohistochemistry. Results The blood levels of glucose, TC and LDL- c were increased significantly in type 2 diabetic group compared with normal control group(P<0. 01). The volume of glomerulus and the deposition of PAS positive substance in the glomerular interstitium were increased under light microscopy, and the glomerular basal membrane was thicker in type 2 diabetic group than those in normal control group under transmission electron microscopy. The mRNA and protein expressions of TGF-β1 and FN were increased(P<0. 01), and the mRNA expression of 11β-HSD2 was decreased(P<0. 05), while the mRNA expression of 11β-HSD1 was unchanged in type 2 diabetic group compared with normal control group. After the treatment of gossypol, the level of the blood glucose was significantly decreased(P< 0. 01), and the levels of TC, LDL-c showed a trend of decrease but had no statistical differences compared with type 2 diabetic group. The morphology of nephridial tissue was ameliorated in gossypol treatment group. The mRNA and protein expressions of TGF-β1 and FN were decreased(0. 16± 0. 02,0. 22±0. 05 ; 0. 24±0. 06,0. 33±0. 07, P< 0. 05), while the mRNA expressions of 11β-HSD1 and 11β-HSD2 were unchanged compared with type 2 diabetic group. Conclusions Gossypol can relieve the pathologic changes of nephridial tissue, inhibit the expressions of TGF-β1 and FN through decreasing blood glucose of rats with diabetes mellitus.     

细胞学标本BRAFV600E检测对常规穿刺活检难以定性诊断的甲状腺结节的应用价值

目的 探讨甲状腺结节常规穿刺活检性质难以明确时,采用相应的细胞学标本BRAFV600E检测的应用价值.方法 回顾性分析2016年3月至2019年3月在丽水市中心医院同时进行甲状腺结节穿刺活检和甲状腺切除手术的患者795例,其中细胞学检测不能明确诊断者135例(TBS 3、4、5类)的细胞学材料进行了BRAFV600E检...     

二巯丙磺钠对大鼠脑缺血再灌注损伤的保护作用

目的 :探讨二巯丙磺钠 (Na DMPS)对大鼠脑缺血再灌注损伤的保护作用。方法 :用复制大鼠 4 动脉阻断模型 ,观察脑缺血再灌注损伤的水份和钙含量变化及组织学和超微结构改变 ,以及Na DMPS脑室内给药 (icv)对上述指标的影响。结果 :脑缺血 2 0min ,再灌注 60min ,可造成脑水份 ,钙含量明显增加 ,组织形态学检查亦见缺血再灌注损伤。缺血前 2 0minicvNa DMPS 90 0 μg·kg 1,能显著降低脑水份和钙含量 ,逆转脑缺血再灌注损伤。结论 :Na DMPS对大鼠脑缺血再灌注损伤有保护作用。     

泛素相关基因在肝细胞肝癌中 的表达及临床意义

目的 探讨泛素相关基因（URGs）在肝细胞肝癌（HCC）中的表达及临床意义。 方法 筛选癌症基因组图谱 （TCGA）数据库中 HCC 患者差异表达的 URGs，以差异表达最明显的泛素结合酶（UBE）2C 为对象，采用 qRT-PCR 法和免疫组 化法验证 UBE2C 的表达及与 HCC 患者临床病理特征、预后的关系；构建与 URGs 相关 HCC 临床预后模型，并绘制 ROC 曲线分 析模型预测患者 1、2、3 年生存期的效能。 结果 qRT-PCR 和免疫组化结果均证实，相比癌旁组织，UBE2C mRNA 和蛋白 在 HCC 组织中均显著高表达（均 P＜0.05）；生存分析结果显示，UBE2C 高表达组累计生存率显著低于 UBE2C 低表达组 （P＜0.01）。TCGA 数据显示 UBE2C 高表达与 HCC 患者年龄及肿瘤分级、分期、大小均显著相关（均 P＜0.01），UBE2C 高 表达是 HCC 患者较差预后的独立影响因素（P＜0.01）。临床数据发现 UBE2C 高表达与肿瘤分级、分期、血清甲胎蛋白 （AFP）及 HBsAg 相关（均 P＜0.05）；TCGA 中发现 11 个 URGs 与 HCC 不良预后相关。构建的预后模型预测患者 1、2 和 3 年生存期的 AUC 分别为 0.762、0.708 和 0.683。 结论 UBE2C 在 HCC 中高表达，且与 HCC 分级、分期、不良预后相关；利用 URGs构建预测HCC的临床预后模型可有效预测患者术后生存期。