目的 探讨在BALB/c小鼠感染日本血吸虫后形成肝纤维化的过程中,转化生长因子-β(transforming growth factorβ,TGF-β)1及其两类受体:TGF-β受体Ⅰ(TβR Ⅰ)、TGF-β受体Ⅱ(TβRⅡ)以及Smad2、Smad3、Smad4和Smad7在转录水平的表达. 方法 50只BALB/c小鼠感染日本血吸虫尾蚴,(20±1)条/只,用于构建肝纤维化模型,10只未感染的BALB/c小鼠作为健康对照组,分别在感染后8、12、16和24周处死小鼠取肝组织,同时取对照组小鼠肝组织.所获肝组织一部分立即液氮冷冻后,保存于-80℃,通过RT-PCR方法测定TGF-β1、TβR Ⅰ、TβRⅡ和Smad2、Smad3、Smad4以及Smad7的mRNA水平,结果为相对值,以待测mRNA密度扫描计数与内参照β-actin mRNA密度扫描计数的比值表示.另一部分肝组织置入常规10％甲醛固定液中,用于伊红-苏木素(HE)染色和天狼猩红染色,其中HE染色用于测定血吸虫虫卵肉芽肿面积(每一个虫卵肉芽肿的最大长度与最大宽度的乘积,以mm2表示),每份标本随机测量5个虫卵肉芽肿面积,求平均值,天狼猩红染色用于判断肝纤维化程度,并以下述方法计分:正常肝组织为0级,以20=1计分;胶原纤维包绕肉芽肿周围并插入其中为Ⅰ级,以21=2计分；汇管区有大量纤维化,小叶间仅有少量纤维为Ⅱ级,以22=4计分;纤维组织大量延伸至小叶间为Ⅲ级,以23=8计分. 结果 小鼠感染日本血吸虫8周后其肝脏中形成的虫卵肉芽肿周围出现胶原纤维,并随着感染时间的延长,胶原纤维逐渐增加.感染后16周,胶原纤维在肝小叶中分布明显,其肝纤维化程度计分为4.27±1.03分;至感染24周时,胶原沉积量达到顶峰,为6.90±1.57分；而正常小鼠肝组织的肝纤维化计分为1分.正常小鼠肝脏中TGF-β1 mRNA的表达水平为0.30±0.18,其表达量在感染后8周达到高峰(0.87±0.76),而后下降,但在感染24周时,其表达量再次升高(1.34±0.52).TβRⅡmRNA在感染8周时有所下降,为0.60±0.30,在感染12周时回升到正常水平,为0.92±0.21,在感染16周时,其表达量又下降为0.76±0.16,而在感染24周时升至1.16±0.73;而正常小鼠肝组织中TβRⅡmRNA的表达水平为1.16±0.25.感染后,Smad2 mRNA在感染12周时和感染24周时均较正常对照(0.85±0.10)有所下降,分别为0.41±0.23和0.50±0.16.Smad3 mRNA在感染16周时有所升高(0.62±0.09),这种高水平表达持续到24周(0.61±0.14).在肝纤维化形成过程中,Smad4 mRNA和Smad7 mRNA以及TβRⅠ mRNA的表达水平与正常对照组比较无明显差异. 结论 在日本血吸虫性肝纤维化形成过程中,下述因子的下调可能诱导肝纤维化形成;TβRⅡmRNA、Smad3 mRNA和处于感染后期的Smad2 mRNA,而Smad7mRNA的正常水平表达在肝纤维化形成中发挥促进作用.在感染早期,Smad2 mRNA表达的下调可能抑制肝纤维化的形成.
目的 探討在BALB/c小鼠感染日本血吸蟲後形成肝纖維化的過程中,轉化生長因子-β(transforming growth factorβ,TGF-β)1及其兩類受體:TGF-β受體Ⅰ(TβR Ⅰ)、TGF-β受體Ⅱ(TβRⅡ)以及Smad2、Smad3、Smad4和Smad7在轉錄水平的錶達. 方法 50隻BALB/c小鼠感染日本血吸蟲尾蚴,(20±1)條/隻,用于構建肝纖維化模型,10隻未感染的BALB/c小鼠作為健康對照組,分彆在感染後8、12、16和24週處死小鼠取肝組織,同時取對照組小鼠肝組織.所穫肝組織一部分立即液氮冷凍後,保存于-80℃,通過RT-PCR方法測定TGF-β1、TβR Ⅰ、TβRⅡ和Smad2、Smad3、Smad4以及Smad7的mRNA水平,結果為相對值,以待測mRNA密度掃描計數與內參照β-actin mRNA密度掃描計數的比值錶示.另一部分肝組織置入常規10％甲醛固定液中,用于伊紅-囌木素(HE)染色和天狼猩紅染色,其中HE染色用于測定血吸蟲蟲卵肉芽腫麵積(每一箇蟲卵肉芽腫的最大長度與最大寬度的乘積,以mm2錶示),每份標本隨機測量5箇蟲卵肉芽腫麵積,求平均值,天狼猩紅染色用于判斷肝纖維化程度,併以下述方法計分:正常肝組織為0級,以20=1計分;膠原纖維包繞肉芽腫週圍併插入其中為Ⅰ級,以21=2計分；彙管區有大量纖維化,小葉間僅有少量纖維為Ⅱ級,以22=4計分;纖維組織大量延伸至小葉間為Ⅲ級,以23=8計分. 結果 小鼠感染日本血吸蟲8週後其肝髒中形成的蟲卵肉芽腫週圍齣現膠原纖維,併隨著感染時間的延長,膠原纖維逐漸增加.感染後16週,膠原纖維在肝小葉中分佈明顯,其肝纖維化程度計分為4.27±1.03分;至感染24週時,膠原沉積量達到頂峰,為6.90±1.57分；而正常小鼠肝組織的肝纖維化計分為1分.正常小鼠肝髒中TGF-β1 mRNA的錶達水平為0.30±0.18,其錶達量在感染後8週達到高峰(0.87±0.76),而後下降,但在感染24週時,其錶達量再次升高(1.34±0.52).TβRⅡmRNA在感染8週時有所下降,為0.60±0.30,在感染12週時迴升到正常水平,為0.92±0.21,在感染16週時,其錶達量又下降為0.76±0.16,而在感染24週時升至1.16±0.73;而正常小鼠肝組織中TβRⅡmRNA的錶達水平為1.16±0.25.感染後,Smad2 mRNA在感染12週時和感染24週時均較正常對照(0.85±0.10)有所下降,分彆為0.41±0.23和0.50±0.16.Smad3 mRNA在感染16週時有所升高(0.62±0.09),這種高水平錶達持續到24週(0.61±0.14).在肝纖維化形成過程中,Smad4 mRNA和Smad7 mRNA以及TβRⅠ mRNA的錶達水平與正常對照組比較無明顯差異. 結論 在日本血吸蟲性肝纖維化形成過程中,下述因子的下調可能誘導肝纖維化形成;TβRⅡmRNA、Smad3 mRNA和處于感染後期的Smad2 mRNA,而Smad7mRNA的正常水平錶達在肝纖維化形成中髮揮促進作用.在感染早期,Smad2 mRNA錶達的下調可能抑製肝纖維化的形成.
목적 탐토재BALB/c소서감염일본혈흡충후형성간섬유화적과정중,전화생장인자-β(transforming growth factorβ,TGF-β)1급기량류수체:TGF-β수체Ⅰ(TβR Ⅰ)、TGF-β수체Ⅱ(TβRⅡ)이급Smad2、Smad3、Smad4화Smad7재전록수평적표체. 방법 50지BALB/c소서감염일본혈흡충미유,(20±1)조/지,용우구건간섬유화모형,10지미감염적BALB/c소서작위건강대조조,분별재감염후8、12、16화24주처사소서취간조직,동시취대조조소서간조직.소획간조직일부분립즉액담냉동후,보존우-80℃,통과RT-PCR방법측정TGF-β1、TβR Ⅰ、TβRⅡ화Smad2、Smad3、Smad4이급Smad7적mRNA수평,결과위상대치,이대측mRNA밀도소묘계수여내삼조β-actin mRNA밀도소묘계수적비치표시.령일부분간조직치입상규10％갑철고정액중,용우이홍-소목소(HE)염색화천랑성홍염색,기중HE염색용우측정혈흡충충란육아종면적(매일개충란육아종적최대장도여최대관도적승적,이mm2표시),매빈표본수궤측량5개충란육아종면적,구평균치,천랑성홍염색용우판단간섬유화정도,병이하술방법계분:정상간조직위0급,이20=1계분;효원섬유포요육아종주위병삽입기중위Ⅰ급,이21=2계분；회관구유대량섬유화,소협간부유소량섬유위Ⅱ급,이22=4계분;섬유조직대량연신지소협간위Ⅲ급,이23=8계분. 결과 소서감염일본혈흡충8주후기간장중형성적충란육아종주위출현효원섬유,병수착감염시간적연장,효원섬유축점증가.감염후16주,효원섬유재간소협중분포명현,기간섬유화정도계분위4.27±1.03분;지감염24주시,효원침적량체도정봉,위6.90±1.57분；이정상소서간조직적간섬유화계분위1분.정상소서간장중TGF-β1 mRNA적표체수평위0.30±0.18,기표체량재감염후8주체도고봉(0.87±0.76),이후하강,단재감염24주시,기표체량재차승고(1.34±0.52).TβRⅡmRNA재감염8주시유소하강,위0.60±0.30,재감염12주시회승도정상수평,위0.92±0.21,재감염16주시,기표체량우하강위0.76±0.16,이재감염24주시승지1.16±0.73;이정상소서간조직중TβRⅡmRNA적표체수평위1.16±0.25.감염후,Smad2 mRNA재감염12주시화감염24주시균교정상대조(0.85±0.10)유소하강,분별위0.41±0.23화0.50±0.16.Smad3 mRNA재감염16주시유소승고(0.62±0.09),저충고수평표체지속도24주(0.61±0.14).재간섬유화형성과정중,Smad4 mRNA화Smad7 mRNA이급TβRⅠ mRNA적표체수평여정상대조조비교무명현차이. 결론 재일본혈흡충성간섬유화형성과정중,하술인자적하조가능유도간섬유화형성;TβRⅡmRNA、Smad3 mRNA화처우감염후기적Smad2 mRNA,이Smad7mRNA적정상수평표체재간섬유화형성중발휘촉진작용.재감염조기,Smad2 mRNA표체적하조가능억제간섬유화적형성.
Objective To investigate the transcription level expression of TGF-β1,its two transmembrane receptors TGF-β receptor Ⅰ (TβRI) and TGF-β receptor Ⅱ (TβR Ⅱ) and Smad2,Smad3,Smad4 and Smad7 during the development of liver fibrosis in the BALB/c mice infected with Schistosomajaponcum.Methods Fifty BALB/c mice infected with cercariae of Schistosomajaponicum (20 ± 1) were used as the liver fibrosis model and 10 uninfected mice belonged to the normal control group.Liver specimens were got at the 8th,12th,16th and 24th week post infection respectively and the normal controls were sacrificed at the same period mentioned above.Some liver tissues were frozen in the fluid nitrogen immediately and then conserved in the-80 ℃ refrigerator for reverse transcription polymerase chain reaction (RT-PCR) to detect the mRNA level of TGF-β1,TβR Ⅰ,TβR Ⅱ,Smad2,Smad3,Smad4 and Smad7.The final value was expressed by the ratio of the scan density of the amplified fragment with the internal control of β-actin,which is a relative value.Other liver pieces were fixed in 10％ buffered formalin for histology assay to detect the size of egg granuloma measured by the product of maximum width and the maximum length and expressed in square micrometers and the final results were the average of the five readings randomly under the microscope and thus to determine the liver fibrosis degree,by the criteria:grade Ⅰ:20=1 no significant collagen was observed in liver tissue; grade Ⅱ:21=2 collagen distributed around and in the granuloma; grade Ⅲ:22=4 more collagen appeared in the portal tracts while few collagen among liver lobules; grade Ⅳ:23=8 fibrous tissues penetrated into liver lobules.Results Collagen fibers appeared around egg granulomas after 8 weeks of Schistosomajaponcum infection and increased gradually.At the 16th week after infection,fibrous tissue distributed evidently in liver lobules and the score of liver fibrosis was 4.27 ± 1.03 and fibrosis degree peaked at the 24th week when scores amounted to 6.90 ± 1.57,while the score of normal mice liver fibrosis was 1.The normal level of TGF-β1 mRNA in liver was 0.30 ± 0.18.It reached the peak 0.87 ± 0.76 at the 8th week,and then decreased,elevated again (1.34 ± 0.52) at the 24th week.TβR Ⅱ mRNA detection demonstrated that reduction (0.60 ± 0.30) at the 8th week,elevation to the normal level 0.92 ± 0.21 at the 12th week,reduction again (0.76 ± 0.16) at 16th week,and increase again (1.16 ± 0.73),at the 24th week compared with the normal level of mRNA of TβR Ⅱ (1.16 ± 0.25).After infection,Smad2 mRNA diminished to 0.41 ± 0.23 and 0.50 ± 0.16 at the 12th and 24th week post-infection,respectively,compared with the normal mRNA level of Smad2 (0.85 ± 0.10) while levels of Smad3 mRNA elevated (0.62 ± 0.09) at the 16th week and remained the higher level (0.61 ± 0.14) till the 24th week.No significant changes were observed in TβRⅠ mRNA,Smad4 mRNA and Smad7 mRNA during the mice liver fibrogensis compared with those in the normal control group.Conclusion In liver fibrogensis induced by Schistosoma japonicum,The down regulation of the following factors may play the induction roles:TβR Ⅱ mRNA,Smad3 mRNA and Smad2 mRNA in latter stage of post-infection,as well as the normal level of Smad7 mRNA may play a positive role in fibrogensis.On the contrary,the reduction of Smad2 mRNA in the early period of post-infection may inhibit liver fibrogensis.
