表現截短依鈣蛋白質分解脢之小次單位對L8肌原細胞依鈣蛋白質分解脢系統的影響

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Title:	表現截短依鈣蛋白質分解脢之小次單位對L8肌原細胞依鈣蛋白質分解脢系統的影響
Other Titles:	Effect of Overexpression of Truncated Calpain Small Subunit on Calpain System in L8 myoblast
Authors:	鍾尊智 Chung, Tsun-Chih
Contributors:	歐柏榮 Ou, Bor-Rung 東海大學畜產與生物科技學系
Keywords:	L8肌原細胞;依鈣蛋白質分解脢系統;截短依鈣蛋白質分解脢之小次單位;表現;蛋白質分解的速度;放射免疫沉澱 calpain;L8 myoblast;Truncated Calpain Small Subunit;Overexpression;radioimmunoprecipitation;protein degradation
Date:	1999
Issue Date:	2011-05-25T09:15:06Z (UTC)
Abstract:	本研究的目的，主要利用分子生物的技術，於肌原細胞內表現經自我分解之活化後截短的依鈣蛋白質分解?之小次單位 ( truncated calpain small subunit; 21KCS )，來瞭解21KCS 對依鈣蛋白質分解?之大次單位 ( calpain large subunit; CL ) 的影響，並期望對肌肉細胞蛋白質分解的機制能有進一步的瞭解。利用已發表的大鼠之依鈣蛋白質分解?之小次單位 ( CS ) 的cDNA序列 ( Graham-Siegenthaler et al., 1994 ) 來設計引子 ( 21K-1; 21K-2 )，用於大鼠肌肉的cDNA基因庫 ( library ) 中，經由聚合?連鎖反應 ( Polymerase Chain Reaction; PCR ) 的方法得到577 bp的21KCS cDNA片段 ( calpain post-autolysis small subunit cDNA; 21Kda; 21KCS )，再將此片段分別植入真核表現質體pMAMneo-SV40 ( pSV )，成為pMAMneo-SV40 small subunit ( pSV-SS )，並利用LIPOFECTAMINETM ( GIBCO, USA ) 的試劑將已構築好的質體pNeo及pSV-SS分別轉殖 ( transfection ) 入大鼠的L8肌原細胞株 ( myoblast )，以G418 ( 300 μg/ ml ) 選殖，進行單株細胞選殖後，得到穩定的轉殖細胞株L8-Neo、SS1、SS2及SS3。將轉殖成功的L8細胞株，萃取其總RNA後，利用引子21K-1及oligo-dT，以反轉錄聚合?連鎖反應法 ( RT-PCR ) 分析，其結果顯示SS2及SS3皆有外源性基因21KCS的表現，SS1則否。為了瞭解表現21KCS對CL的影響，分別萃取轉殖細胞之細胞質蛋白質及膜蛋白質且定量之，再用μCL 及mCL ( 80Kda ) 的專一抗體 ( Hong et al., 1995 ) 以西方吸漬法來探測依鈣蛋白質分解?於L8肌原細胞中表現的情形。結果顯示，將L8轉殖細胞株與對照組L8-Neo相比較後發現，SS2與SS3在細胞質中μCL分別增加15.7 %及17.3 %；在mCL分別增加23.3 %及16.6 %，但SS1則皆無顯著增加。膜蛋白質上的μCL及mCL並無顯著差異。此外，為瞭解表現21KCS對依鈣蛋白質分解?系統穩定度的影響，利用放射免疫沉澱 ( radioimmunoprecipitation ) 分析法測量L8轉殖細胞株SS3與對照組L8-Neo的穩定度 ( stability )，結果顯示L8轉殖細胞株SS3與對照組L8-Neo在 μ-calpain分別為36.3小時及31.9小時；在m-calpain分別為40.5小時及36.3小時，顯示在穩定度上分別增加4.4小時及4.2小時。為瞭解細胞質中 μ-及m-calpain增加後對細胞蛋白質的分解是否有影響，以3H-tyrosine標示蛋白質的方法測量各轉殖細胞株蛋白質的分解速度，結果顯示轉殖細胞株SS1、SS2及SS3與對照組間的總蛋白質分解速率並無顯著差異。總合上述，表現21KCS於大鼠L8肌原細胞會導致L8轉殖細胞中?CL及mCL的量顯著增加，此增加的現象乃因?CL及mCL的穩定度增加所累積的。細胞內依鈣蛋白質分解?量的增加並不會影響其移位到細胞膜的量，因此不會增加依鈣蛋白質分解?活化的量和細胞內蛋白質分解的速度。 The growth rate of skeletal muscle dependents on the rates of muscle protein synthesis and protein degradation. It was reported that calpains played an important role on the initiation of muscle protein degradation. The calpains depend on its calcium requirement can be grouped into ?- and m-calpain. Both ??and m-calpain contain two subunit, small subunit ( CS; 30KDa ) and large subunit ( CL; 80KDa ). The objectives of this study were to investigate the function of CS by expressing the autolysis form of CS in L8 myoblast. Rat post-autolysis small subunit ( 21KCS; 21KDa ) cDNA was amplified by polymerase chain reaction ( PCR ). The cDNA ( 577bp ) then was inserted into pMAMneo-SV40 ( pSV ) vector. The new plasmid is referred to pMAMneo-SV40-Small subunit ( pSV-SS ). The plasmid pSV and pSV-SS were transfected into L8 myoblasts, respectively. Then the single cloning was selected by G418 ( 300?g/ ml ) containing medium. After selection, the transfected cell were analyzed by PCR using Neor primer against the genomic DNA and stable cloning cell line L8-Neo, SS1, SS2 and SS3 were established. CS mRNA were analyzed by Reverse transcription polymerase chain reaction ( RT-PCR ) in transfected and control cells using primer 21K-1 and oligo-dT. The data indicated that SS2 and SS3 had expression of exogenous CS mRNA ( 21KCS mRNA ), however, SS1 did not express the exogenous CS mRNA. In addition, ??and m-calpain were analyzed by Western blot in total and membrane protein. In analysis of total protein, the results showed that the protein concentration of ?- calpain in SS2 and SS3 were 15.7% and 17.3% higher than control ( L8-Neo ), respectively. The protein concentration of m-calpain in SS2 and SS3 were 23.3% and 16.6% higher than control ( L8-Neo ), respectively. In addition, SS3 and control ( L8-Neo ) were analyzed by immunoprecipitation to detect the stability of calpain. The data indicated that the stability of ?-calpain in SS3 was 36.3 hours higher than control ( 31.9hours ). The stability of ?-calpain was 40.5 hours higher than control ( 36.3hours ). In analysis of membrane calpain concentration, the transfected cells ( SS1、SS2 and SS3 ) and control cell ( L8-Neo ) have no significant difference by expression of exogenous CS. Also, there is no different in total protein degradation between transfected cells and control cells. These results imply that expression of exogenous 21KCS can increase the protein level of ?- and m-calpain and increase the stability of calpains. The result indicated that 21KCS effect on translation level but not on transcription level. On the pther hand, 21KCS can not improve the opportunity to translocte to the membrane or change the rate of protein degradation in myoblast of rat.
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