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£ÛÕªÒª£ÝÄ¿µÄ£ºÌ½Ë÷°ÐÏòbcl-2±àÂë»ùÒòµÄshRNA±í´ïÔØÌåÒÖÖÆ¸ß±í´ïµÄ bcl-2µ°°×£¬ÓÕµ¼HepG-2ϸ°ûµòÍö¡¢ÒÖÖÆÆäÉú³¤µÄ×÷Óã¬ÒÔÆÚΪÔËÓÃRNAi¼¼ÊõÖÎÁÆÖ×ÁöÌṩÀíÂÛ»ù´¡ºÍʵÑéÒÀ¾Ý¡£·½·¨£º²ÉÓÃDNAÖØ×é¡¢RT-PCR¡¢DNAÐòÁвⶨ¡¢Ï¸°ûµòÍöÓëÔöÖ³¼ì²âµÈ¼¼Êõ³õ²½¹Û²ìÁ˰ÐÏòbcl-2±àÂë»ùÒòµÄshRNA±í´ïÔØÌåÒÖÖÆbcl-2»ùÒòת¼Óë·Ò룻ÓÕµ¼¸ß±í´ïbcl-2»ùÒòµÄHepG-2ϸ°ûµòÍö¼°ÔöÖ³ÊÜÒÖ×÷Ó᣽á¹û£º·ÖÎö±È½Ï֤ʵ±¾Îijɹ¦¹¹½¨µÄ3¸ö°ÐÏòbcl-2µÄshRNA±í´ïÔØÌåÖ®Ò»£¬¼´pGenesil-B2¿ÉÓÐЧÒÖÖÆbcl-2±í´ï£¨mRNAת¼ÒÖÖÆÂÊΪ49.1%£©¡¢²¢ÖÂϸ°ûµòÍö£¨Ï¸°ûµòÍöÂÊΪ65.12%£©ºÍÔöÖ³ÒÖÖÆ£¨ÒÖÖÆÂÊΪ46.4%£©¡£ÕâЩÑо¿½á¹û±íÃ÷ÄÜÓÐЧÒÖÖÆÖ×Áöϸ°û¸ß±í´ïµÄbcl-2»ùÒòµÄRNA¸ÉÈż¼Êõ£¬ÓпÉÄÜÓÃÓÚÁÙ´²Ö×ÁöÖÎÁÆ¡£ ¹Ø¼ü´Ê£ºÔöÇ¿ÐÍÂÌɫӫ¹âµ°°× , Bϸ°û°×Ѫ²¡-2 ,¶Ì·¢¼ÐRNA , RNA¸ÉÈÅ£¬¸Î°©Ï¸°ûϵ¡£ THE INHIBITING OF shRNA ON BCL-2 EXPRESSION IN HEPG-2 CELL objective: to observe the inhibition of shRNA targeting bcl-2 gene on the expression of bcl-2 in HepG-2 cells. Methods: The(Three couple) DNA sequence targeting bcl-2 gene was cloned into the pGenesil-1, which is the vector expressing shRNA and enhance green fluorescent protein(EGFP) in mammalian cells, the recombinant vector was confirmed with restriction enzyme digestion¡¢PCR and sequencing. Then RT-PCR was used to observe the recombinant vectors on inhibiting the genetic transcription and translation of bcl-2; The technique for detecting apoptosis and generation was used to observe the effects of the recombinant vectors inducing apoptosis and inhibiting generation for the HepG-2 cell expressing bcl-2 .Results: The recombinant vectors expressing shRNA were constructed successfully. Only one of the three vector expressing shRNA targeting bcl-2 could inhibit the HepG-2 expression of bcl-2(inhibition ratio of mRNA was 49.1%, induce the HepG-2 apoptosis ratio was 65.12% and inhibit HepG-2 generation ratio was 46.4%. So It is feasible to use RNAi to inhibit the replication of tumor cell and may be effective for the cancer therapy. Key words: EGFP, bcl-2, shRNA, RNAi, HepG-2 Ö×ÁöÊÇÑÏÖØÍþвÈËÀཡ¿µµÄ¼²²¡Ö®Ò»£¬Æä·¢²¡ÂʳÊÉÏÉýÇ÷ÊÆ¡£ÈçºÎÓÐЧÖÎÁƺͿØÖÆÖ×ÁöÒ»Ö±ÊÇҽѧÁìÓò¡¢ÄËÖÁÉúÃü¿ÆÑ§ÁìÓò¼±´ý½â¾öµÄÖØ´óÄÑÌâ[1]¡£bcl-2ÊÇÒ»ÖÖϸ°ûµòÍöÒÖÖÆ»ùÒò£¬¹ý¶È±í´ïbcl-2µÄת»ùÒòСÊó¿É·¢ÉúÖ×Áö[2]£» bcl-2»ùÒòÒ×λ¡¢»î»¯¿É¼ûÓÚ¶àÖÖBϸ°ûÁܰÍÁö¡£ÕâЩÑо¿½á¹ûÌáʾbcl-2¿ÉÄÜÊÇÒ»ÖÖÄÜͨ¹ý×è¶Ïϸ°ûµòÍö×÷Ó㬶øÖÂÖ×Áö·¢ÉúµÄÖØÒª·Ö×Ó£¬²¢ÇÒÔÚÖ×Áö¶Ô»¯ÁÆÒ©ÎïµÄ·´Ó¦ÐÔÓ뿹ҩÐÔÖУ¬bcl-2¼°ÆäÏà¹Ø»ùÒòÆð×ÅÖØÒªµÄµ÷¿Ø×÷ÓÃ[3¡¢4]¡£¾Ý´ËÉèÏëÌØÒìÐÔ·â±ÕºÍ×è¶ÏÖ×Áöϸ°ûµÄbcl-2»ùÒò±í´ï£¬ÓпÉÄÜÒÖÖÆÖ×Áöϸ°ûÔöÖ³¡£ RNA¸ÉÈÅ(RNA interference£¬RNAi)ÊÇͨ¹ýÄÚ¡¢ÍâÔ´ÐÔСÐÍË«Á´¸ÉÈÅRNA(short/small interference RNA£¬ siRNA)°ÐÏòÓÕµ¼mRNAˮƽÉϵĻùÒò³ÁĬ £¬ÌØÒìÐÔÒÖÖÆÏ¸°ûÄÚͬԴ»ùÒò±í´ïºÍµ°°×ÖʺϳɵĹý³Ì£¬Êǹ㷺´æÔÚÓÚ¶¯Ö²ÎïÖеÄÒ»ÖÖ»ùÒò³ÁĬ·½Ê½[5¡¢6]¡£siRNAÔÚ²¸È鶯Îïϸ°ûÖв»½ö¿ÉÒÔÓÕµ¼Ç¿ÓÐÁ¦µÄÌØÒìÐÔ RNAi×÷Ó㬶øÇÒÒ²¿ÉÒÔÕë¶Ô¶à¸ö»ùÒò»ò»ùÒò×åµÄ¹²ÓÐÐòÁÐÀ´ÒÖÖÆ¶à¸ö»ùÒò±í´ïµÄÑо¿½á¹û±íÃ÷ÆäÓпÉÄܳÉΪ¾ß¹ã·ºÓ¦ÓÃǰ¾°µÄ¿¹Ö×ÁöµÄ»ùÒòÖÎÁÆ·½·¨[7]¡£ÕâÒ»¿ÉÓëÈËÀà»ùÒò×鼯»®£¨human genomics program£¬HGP£©ÏàÌá²¢ÂÛµÄÍ»ÆÆÐÔÑо¿½øÕ¹¿ª´´ÁËÈËÀàÖ×Áö»ùÒòÖÎÁƵÄÐÂÌìµØ£¬ÎªÖ×ÁöÖÎÁÆÌṩÁËÐÂÏßË÷¡¢ÐÂ˼·ºÍз½·¨[8]¡£Ñо¿ÒÔpGenesil-1ÔØÌåΪ¹Ç¼Ü£¬¹¹½¨ÁË´øÔöÇ¿ÐÍÂÌɫӫ¹âµ°°×£¨enhance green fluorescent protein£¬EGFP £©±¨¸æ»ùÒòµÄ¡¢°ÐÏòbcl-2»ùÒòµÄshRNA±í´ïÔØÌ壻ÒԸ߱í´ïbcl-2 »ùÒòµÄHepG-2ϸ°ûΪ°Ðϸ°û£¬Ì½Ë÷Á˰ÐÏòbcl-2µÄshRNA±í´ïÔØÌåÒÖÖÆ¸Ãϸ°ûbcl-2±í´ïµÄ¡¢ÓÕµ¼¸Ãϸ°ûµòÍöºÍÉú³¤ÒÖÖÆµÄ×÷Óã¬ÎªÀûÓÃRNAi¼¼ÊõÖÎÁÆÁÙ´²Ö×ÁöÌṩÁËÀíÂÛ»ù´¡ºÍʵÑéÒÀ¾Ý¡£ 1×ÊÁÏÓë·½·¨ 1.1 Ö÷ÒªÊÔ¼Á Я´øEGFP»ùÒòµÄpGenesil-1£¨Î人¾§ÈüÉúÎï¼¼Êõ¹«Ë¾£©¡¢Ð¡Á¿ÖÊÁ£ÌáÈ¡ÊÔ¼ÁºÐ£¨ÉϺ£»ªË´ÉúÎïÓÐÏÞ¹«Ë¾£©£¬T4DNAÁ¬½Óø¡¢ÏÞÖÆÐÔÄÚÇÐøµÈ£¨MBI¹«Ë¾£©¡¢×ªÈ¾ÊÔ¼Álipofectamine 2000£¨invitrogen£©¡¢Êó¿¹ÈËbcl-2µ¥¿¹£¨Ancell Corporation£©¡¢HRP±ê¼ÇµÄÑò¿¹Êó¦ÊÁ´£¨µ¥¿¹£©£¨Southern Biotechnology Associates, Inc£©¡¢Ï¸°ûµòÍö¼ì²âÊÔ¼ÁºÐ£¨BioVision, Inc£©µÈ¡£ 1.2 °ÐÏòbcl-2»ùÒò¹ÑºËÜÕËáµÄÉè¼Æ ¸ù¾ÝshRNAÉè¼ÆÔÔòºÍGenBankÖеÄbcl-2±àÂëÐòÁУ¬Ñ¡ÔñÉè¼ÆµÄÈý¶Ô°ÐÏòbcl-2»ùÒòµÄDNAÐòÁÐ (·Ö±ðÃüÃûΪB1¡«B3) ºÍÒ»¶ÔÓëÈκÎÈËÀà»ùÒòÐòÁоùÎÞͬԴÐÔµÄDNAÐòÁÐ×÷ΪÒõÐÔ¶ÔÕÕÐòÁУ¨ÃüÃûΪS£©£¬ÔÚÆäÁ½¶ËÌí¼ÓBgl¢òºÍHind¢ó øÇÐλµã£¬ÓÃTTTTT×÷Ϊת¼ÖÕÖ¹×Ó£¬²¢ÓÃBLASTÈí¼þ½øÐÐͬԴÐÔ·ÖÎö¡£¸÷¶ÔsiRNAÐòÁÐÈçÏ£º B1£ºÕýÒåÁ´£º5¡ä-GAT CTG CTG CAC CTG ACG CCC TTC TTC AAG AGA GAA GGG CGT CAG GTG CAG CTT TTT-3¡ä¡¢·´ÒåÁ´5¡ä-AGC TTA AAA AGC TGC ACC TGA CGC CCT TCT CTC TTG AAG AAG GGC GTC AGG TGC AGC-3¡ä £» B2£ºÕýÒåÁ´£º5¡ä- GAT CTG AAG TAC ATC CAT TAT AAG TTC AAG AGA CTT ATA ATG GAT GTA CTT CTT TTT-3¡ä¡¢·´ÒåÁ´£º3¡ä-AGC TTA AAA AGA AGT ACA TCC ATT ATA AGT CTC TTG AAC TTA TAA TGG ATG TAC TTC-3¡ä;B3£ºÕýÒåÁ´3¡ä-GAT CTG GGA GAT AGT GAT GAA GTA TTC AAG AGA TAC TTCATC ACT ATC TCC CTT TTT-3¡ä¡¢·´ÒåÁ´5¡ä-AGC TTA AAA AGG GAG ATA GTG ATG AAG TAT CTC TTG AAT ACT TCA TCA CTA TCT CCC-3¡ä;ÒõÐÔ¶ÔÕÕÐòÁÐS£ºÕýÒåÁ´£ºÐòÁÐΪ5¡ä-GAT CTC CTT AGA GTC TCC TGA GCT TCA AGA GAG CTC AGG AGA CTC TAA GGC TTTTT-3¡ä¡¢·´ÒåÁ´£º3¡ä-AGC TTA AAA GCC TTA GAG TCT CCT GAG CTC TCT TGA AGC TCA GGA GAC TCT AAG GC-3¡ä¡£ 1.3 bcl-2 shRNA±í´ïÔØÌåµÄ¹¹½¨ ½«ÉÏÊöºÏ³ÉµÄ¹ÑºËÜÕËá·Ö±ðÈܽâÓÚ100¦ÌlÎÞ¾úË«ÕôË®ÖУ¬È¡ÏàÓ¦µÄÕý·´ÒåÁ´¸÷5¦Ìl,¼ÓÈ뺬5¦Ìl 100mmolNaclµÄEP¹ÜÖУ¬»ìÔÈ£¬ÓÚ94¡æË®Ô¡ 5minºó£¬´ýÆä×ÔÈ»ÀäÈ´£¬Ê¹±àÂëshRNA µÄÁ½Ìõ¹ÑÍÑÑõºËÌǺËËáÍË»ð£»ÓÃT4 DNAÁ¬½Óø½«ÒѸ´ÐԵĸ÷¶ÔDNAÐòÁзֱð²åÈëÓÃBamH IºÍHind ¢ó˫øÇеÄpGenesil-1ÖÊÁ£ÖУ¬Á¬½Ó²úÎïת»¯¸ÐÊÜ̬DH5a¡£´Óת»¯µÄDH5aÖÐÌáÈ¡ÖØ×éÖÊÁ££¬ÓÃBgl¢òºÍHind ¢ó øÇÐÖØ×éÖÊÁ£¡¢ 1£¥µÄÇíÖ¬ÌÇÄý½ºµçÓ¾¼ø¶¨£¬×îºó½«¾Ã¸Çк͵çÓ¾·ÖÎöÈ·ÈϵÄÑôÐÔÖØ×éÖÊÁ£ËÍÉϺ£»ù¿µÉúÎ﹤³ÌÓÐÏÞ¹«Ë¾½øÐÐDNAÐòÁвⶨ¡£¾ÉÏÊö·½·¨¼ø¶¨È·ÈϵİÐÏòbcl-2»ùÒòµÄÈý¸öÑôÐÔÖØ×éÖÊÁ£±»ÒÀ´ÎÃüÃûΪpGenesil-B1¡«B3; ÒõÐÔ¶ÔÕÕÖØ×éÖÊÁ£±»ÃüÃûΪpGenesil-S¡£ 1.4 HepG-2ϸ°ûµÄÅàÑøÓë´«´ú HepG-2ϸ°ûÅàÑøÓÚ10%ÐÂÉúţѪÇå(FCS)¡¢100 u£¯mLÇàÃ¹ËØ¡¢100ug£¯mlÁ´Ã¹ËصÄRPMI1640ÍêÈ«ÅàÑøÒºÖУ¬ÖÃ37¡æ ¡¢5£¥ CO2±¥ºÍʪ¶ÈÅàÑøÏäÖÐÅàÑø¡£µ¹ÖÃÏÔ΢¾µÏ¹۲ìϸ°ûÉú³¤Çé¿ö£¬2¡«3d´«´ú1´Î¡£ËùÓÐʵÑé¾ùÑ¡ÓöÔÊýÉú³¤ÆÚϸ°û¡£ 1£®5 bcl-2 mRNA°ë¶¨Á¿·ÖÎöRT-PCR 1.5.1 ϸ°ûתȾ
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