本研究旨在探讨肝复胶囊(Gan Fu capsule, GFC)对TGF-1诱导HSC-T6细胞增殖的影响及作用机制。试验采用制备空白血清和GFC含药血清,体外培养HSC-T6细胞24 h后,CCK8法筛选最佳给药血清浓度,采用不同浓度TGF-1诱导HSC-T6细胞,CCK8法筛选最...本研究旨在探讨肝复胶囊(Gan Fu capsule, GFC)对TGF-1诱导HSC-T6细胞增殖的影响及作用机制。试验采用制备空白血清和GFC含药血清,体外培养HSC-T6细胞24 h后,CCK8法筛选最佳给药血清浓度,采用不同浓度TGF-1诱导HSC-T6细胞,CCK8法筛选最佳造模浓度,将细胞分为空白组(CON)、模型组(MOD)、肝复胶囊低剂量组(GFC-L)、肝复胶囊中剂量组(GFC-M)、肝复胶囊高剂量组(GFC-H),采用细胞增殖检测(CCK8)法检测各组细胞增殖情况,酶联免疫吸附试验(ELISA)检测IL-6、IL-1 、TNF-含量,蛋白免疫印记试验(Western blot)检测各细胞中核苷酸结合寡聚化结构域样受体蛋白3(Nod-like receptor protein 3,NLRP3)、凋亡相关斑点样蛋白(Apoptosis associated speck-like protein containing a caspase activating recruitment domain, ASC)、半胱氨酸蛋白酶-1(Cysteinyl aspartate specific proteinase-1,Caspase-1)及-平滑肌动蛋白(Alpha-smooth muscle actin,-SMA)的表达。结果表明,与CON组相比,GFC能抑制HSC-T6细胞的增殖能力,减少HSC分泌IL-6、IL-1 、TNF-含量,降低HSC中NLRP3、ASC、Caspase-1及-SMA的表达量。GFC可能通过调节NLRP3/ASC/Caspase-1信号通路和减少-SMA蛋白表达,从而抑制HSC-T6增殖,起到抗肝纤维化的作用。展开更多
In order to elucidate the molecular mechanisms of the oyster (Crassostrea ariakensis) against adverse stimulating factors, we cloned and sequenced a partial cDNA encoding a 70 kDa heat shock cognate protein (Hsc70) fr...In order to elucidate the molecular mechanisms of the oyster (Crassostrea ariakensis) against adverse stimulating factors, we cloned and sequenced a partial cDNA encoding a 70 kDa heat shock cognate protein (Hsc70) from the oyster. The live oysters were obtained from Chengcun, Yangxi County, Guangdong Province, China. Various tissues, including mantle, gills, adductor muscle, heart and blood cells, were respectively collected from 5 untreated live oysters or treated ones at 36℃ for 1 5 hours, and immediately frozen in liquid nitrogen except for the blood cells which were suspended with Trizol Reagent after centrifugation ( 12 000 r/min for 30 s) and stored at -20℃. Total RNA was isolated using Trizol Reagent according to the manufacture’s instructions. The first strand cDNA was synthesized using reverse transcriptase Superscript Ⅱ according to the manufacture’s instructions. The primers were designed from a conserved region of C. gigas Hsc70 cDNA sequence (GeneBank accession No. AF144646). The polymerase chain reaction (PCR) was performed for 30 cycles with denaturation at 94℃ for 30 s, annealing at 49℃ for 40 s, and elongation at 72℃ for 30 s. The product was cloned to pGEM T easy vector and sequenced. It is 509 base pairs (bp) and possesses 94% identity with the cDNA encoding C. gigas Hsc70 using Blastn. This homology was strongly confirmed by amino acid sequence comparison using the Blastx (99%). The 509 bp fragment was labeled with α 32 pdCTP and a random primer DNA labeling kit and employed as a probe to perform Southern blotting, the result demonstrated that the cDNA came from a partial mRNA transcript of C. ariakensis genomic DNA gene. The polymerase chain reaction (PCR) was carried out to investigate the expression of Hsc70, Using the cDNAs of several tissues, such as gills (heat shocked), mantle, adductor muscle (heat shocked), heart, blood cells (one sample with heat shock for 1 5 hours at 36℃ and another without any stimulus). The PCR results revealed that Hsc70 transcripts could be detected in all the tissues analyzed and greatly increased in the tissues with heat shock. The results showed that the Hsc70 is ubiquitously and constitutively expressed but can be stimulated by heat shock. All the facts above firmly established that the cloned cDNA fragment was a part of the cDNA encoding a Hsc70 protein in the oyster C. ariakensis .展开更多
目的观察三甲益肝颗粒(San Jia Yi Gan granula,SJG)含药血清对HSC-T6细胞形态、增殖和凋亡的作用,探讨SJG抗肝纤维化的细胞学基础。方法雄性SD大鼠30只按随机数字表法分为5组,每组6只,分别为生理盐水对照组(Control)、水飞蓟宾组(Sily...目的观察三甲益肝颗粒(San Jia Yi Gan granula,SJG)含药血清对HSC-T6细胞形态、增殖和凋亡的作用,探讨SJG抗肝纤维化的细胞学基础。方法雄性SD大鼠30只按随机数字表法分为5组,每组6只,分别为生理盐水对照组(Control)、水飞蓟宾组(Sily)、低剂量SJG组(SJG-L)、中剂量SJG组(SJG-M)和高剂量SJG组(SJG-H)。SJG按低、中、高(1.7、3.4、6.8g/kg)3个剂量灌胃大鼠制备含药血清,并用不同体积分数(5%、10%、20%、40%)SJG含药血清干预HSC-T6细胞株,用倒置相差显微镜观察HSC-T6细胞形态学的变化,MTT法检测SJG药物血清对HSC-T6细胞增殖的影响,流式细胞仪检测SJG药物血清诱导HSC-T6细胞早期凋亡。结果含药血清干预后,细胞形态发生明显改变:药物组细胞较对照组细胞明显减少,细胞间隙较宽,细胞皱缩,核染色质浓集呈球形。MTT实验结果显示各组药物血清均可抑制HSC-T6细胞的增殖,SJG-M、SJG-H组对HSC-T6细胞增殖的抑制均显著高于Sily组(P<0.05),SJG-L组与Sily组对HSC-T6细胞增殖的抑制无显著差异(P>0.05)。SJG-M、SJG-H组凋亡率分别为(7.09±1.04)%、(11.63±1.53)%,显著高于Sily组[(4.38±1.07)%]、SJG-L组[(4.17±0.30)%]和对照组[(1.74±0.11)%]。与对照组比较,各药物血清干预组细胞凋亡数明显增加,以SJG-H组增加最为明显。结论SJG含药血清可显著抑制HSC-T6细胞的活化、增殖,并诱导其凋亡。展开更多
目的观察TGF-β1对大鼠肝星状细胞系HSC-T6增殖、细胞周期以及分泌肝纤维化相关胶原的影响。方法采用MTT法检测细胞增殖;使用流式细胞仪检测细胞周期;采用荧光定量RT-PCR法检测SMAD3、c-myc、cdk-2、cyclinE、EGF、HGF、Bcl-2、NF-κB、...目的观察TGF-β1对大鼠肝星状细胞系HSC-T6增殖、细胞周期以及分泌肝纤维化相关胶原的影响。方法采用MTT法检测细胞增殖;使用流式细胞仪检测细胞周期;采用荧光定量RT-PCR法检测SMAD3、c-myc、cdk-2、cyclinE、EGF、HGF、Bcl-2、NF-κB、MMP1、MMP9、MMP14、TIMP-1、PAI-1、α-SMA、COLⅠ及COLⅢ等基因mRNA水平;采用ELISA法检测细胞培养液COLⅠ、COLⅢ和α-SMA含量。结果与对照组比,TGF-β1处理24h及36h时,细胞形态及生长状况均无明显变化;在24h和36h时,TGF-β1处理组细胞G0/G1期细胞比率均减少(24h:57.3±8.5%vs 60.6±9.7%;36h:53.0±2.2%vs 56.6±5.0%),S期细胞比例略高(24h:30.6±7.2%vs26.4±10.1%;36h:35.2±3.7%vs 30.8±2.5%),但差异无统计学意义(P>0.05);TGF-β1处理组SMAD3、c-myc、cdk2、cyclin E、EGF、Bcl-2、NF-κB、TIMP1、PAI-1、α-SMA及COLⅠmRNA在24h和36h表达均上调,HGF、MMP1、MMP9、MMP14及COLⅢmRNA在24h时表达下调,36h时表达上调;TGF-β1处理组HSC-T6细胞分泌COLⅠ[24h:(63.0±7.4ng/ml vs 33.2±10.8 ng/ml,P<0.05;36h:58.5±6.0ng/ml vs 42.2±6.3ng/ml,P<0.05];α-SMA[24h:20.6±2.6ng/ml vs 4.2±0.7ng/ml,P<0.05;36h:59.7±14.6ng/ml vs 36.8±5.6ng/ml,P<0.05)均显著增加。结论 TGF-β1对大鼠肝星状细胞系HSC-T6显示了促增殖作用,并能促进肝纤维化相关胶原的分泌。展开更多
文摘In order to elucidate the molecular mechanisms of the oyster (Crassostrea ariakensis) against adverse stimulating factors, we cloned and sequenced a partial cDNA encoding a 70 kDa heat shock cognate protein (Hsc70) from the oyster. The live oysters were obtained from Chengcun, Yangxi County, Guangdong Province, China. Various tissues, including mantle, gills, adductor muscle, heart and blood cells, were respectively collected from 5 untreated live oysters or treated ones at 36℃ for 1 5 hours, and immediately frozen in liquid nitrogen except for the blood cells which were suspended with Trizol Reagent after centrifugation ( 12 000 r/min for 30 s) and stored at -20℃. Total RNA was isolated using Trizol Reagent according to the manufacture’s instructions. The first strand cDNA was synthesized using reverse transcriptase Superscript Ⅱ according to the manufacture’s instructions. The primers were designed from a conserved region of C. gigas Hsc70 cDNA sequence (GeneBank accession No. AF144646). The polymerase chain reaction (PCR) was performed for 30 cycles with denaturation at 94℃ for 30 s, annealing at 49℃ for 40 s, and elongation at 72℃ for 30 s. The product was cloned to pGEM T easy vector and sequenced. It is 509 base pairs (bp) and possesses 94% identity with the cDNA encoding C. gigas Hsc70 using Blastn. This homology was strongly confirmed by amino acid sequence comparison using the Blastx (99%). The 509 bp fragment was labeled with α 32 pdCTP and a random primer DNA labeling kit and employed as a probe to perform Southern blotting, the result demonstrated that the cDNA came from a partial mRNA transcript of C. ariakensis genomic DNA gene. The polymerase chain reaction (PCR) was carried out to investigate the expression of Hsc70, Using the cDNAs of several tissues, such as gills (heat shocked), mantle, adductor muscle (heat shocked), heart, blood cells (one sample with heat shock for 1 5 hours at 36℃ and another without any stimulus). The PCR results revealed that Hsc70 transcripts could be detected in all the tissues analyzed and greatly increased in the tissues with heat shock. The results showed that the Hsc70 is ubiquitously and constitutively expressed but can be stimulated by heat shock. All the facts above firmly established that the cloned cDNA fragment was a part of the cDNA encoding a Hsc70 protein in the oyster C. ariakensis .
文摘目的观察三甲益肝颗粒(San Jia Yi Gan granula,SJG)含药血清对HSC-T6细胞形态、增殖和凋亡的作用,探讨SJG抗肝纤维化的细胞学基础。方法雄性SD大鼠30只按随机数字表法分为5组,每组6只,分别为生理盐水对照组(Control)、水飞蓟宾组(Sily)、低剂量SJG组(SJG-L)、中剂量SJG组(SJG-M)和高剂量SJG组(SJG-H)。SJG按低、中、高(1.7、3.4、6.8g/kg)3个剂量灌胃大鼠制备含药血清,并用不同体积分数(5%、10%、20%、40%)SJG含药血清干预HSC-T6细胞株,用倒置相差显微镜观察HSC-T6细胞形态学的变化,MTT法检测SJG药物血清对HSC-T6细胞增殖的影响,流式细胞仪检测SJG药物血清诱导HSC-T6细胞早期凋亡。结果含药血清干预后,细胞形态发生明显改变:药物组细胞较对照组细胞明显减少,细胞间隙较宽,细胞皱缩,核染色质浓集呈球形。MTT实验结果显示各组药物血清均可抑制HSC-T6细胞的增殖,SJG-M、SJG-H组对HSC-T6细胞增殖的抑制均显著高于Sily组(P<0.05),SJG-L组与Sily组对HSC-T6细胞增殖的抑制无显著差异(P>0.05)。SJG-M、SJG-H组凋亡率分别为(7.09±1.04)%、(11.63±1.53)%,显著高于Sily组[(4.38±1.07)%]、SJG-L组[(4.17±0.30)%]和对照组[(1.74±0.11)%]。与对照组比较,各药物血清干预组细胞凋亡数明显增加,以SJG-H组增加最为明显。结论SJG含药血清可显著抑制HSC-T6细胞的活化、增殖,并诱导其凋亡。
文摘目的观察TGF-β1对大鼠肝星状细胞系HSC-T6增殖、细胞周期以及分泌肝纤维化相关胶原的影响。方法采用MTT法检测细胞增殖;使用流式细胞仪检测细胞周期;采用荧光定量RT-PCR法检测SMAD3、c-myc、cdk-2、cyclinE、EGF、HGF、Bcl-2、NF-κB、MMP1、MMP9、MMP14、TIMP-1、PAI-1、α-SMA、COLⅠ及COLⅢ等基因mRNA水平;采用ELISA法检测细胞培养液COLⅠ、COLⅢ和α-SMA含量。结果与对照组比,TGF-β1处理24h及36h时,细胞形态及生长状况均无明显变化;在24h和36h时,TGF-β1处理组细胞G0/G1期细胞比率均减少(24h:57.3±8.5%vs 60.6±9.7%;36h:53.0±2.2%vs 56.6±5.0%),S期细胞比例略高(24h:30.6±7.2%vs26.4±10.1%;36h:35.2±3.7%vs 30.8±2.5%),但差异无统计学意义(P>0.05);TGF-β1处理组SMAD3、c-myc、cdk2、cyclin E、EGF、Bcl-2、NF-κB、TIMP1、PAI-1、α-SMA及COLⅠmRNA在24h和36h表达均上调,HGF、MMP1、MMP9、MMP14及COLⅢmRNA在24h时表达下调,36h时表达上调;TGF-β1处理组HSC-T6细胞分泌COLⅠ[24h:(63.0±7.4ng/ml vs 33.2±10.8 ng/ml,P<0.05;36h:58.5±6.0ng/ml vs 42.2±6.3ng/ml,P<0.05];α-SMA[24h:20.6±2.6ng/ml vs 4.2±0.7ng/ml,P<0.05;36h:59.7±14.6ng/ml vs 36.8±5.6ng/ml,P<0.05)均显著增加。结论 TGF-β1对大鼠肝星状细胞系HSC-T6显示了促增殖作用,并能促进肝纤维化相关胶原的分泌。
