| The occurrence and development of leukemia is a multi step processor, which is affected by many factors, and involved in leukemic cell over-proliferation, and the blocked differentiation and apoptosis. In physiological conditions, cell proliferation, differentiation, maturation and apoptosis are strickly controlled by a variety of positive and negative regulators, they keep in a dynamic balance, maintaining the stability of blood environment. One mechanism of leukemia pathogenesis is negative feedback inhibitor of reactive cells occurs change it to escape normal growth control. TGF-βis one of the most important negative regulatory factors, TGF-βsignal transduction pathway has an important role in the regulation of cell proliferation differentiation and apoptosis.TGF-βreceptor has three subtypes, such as TGF-βRI, TGF-βRII and TGF-βRⅢ. TGF-βRII has a high affinity to and TGF-β, which plays a important role in the TGF-β-mediated signal transduction. TGF-βRⅡwere found abnormal expressed in many tumors,In pancreatic cancer and gallbladder cancer, the expression of TGF-βRⅡdecreased,In colon cancer ,the mutation of TGF-βRⅡled to the tumor cells lose TGF-βsensitivity. To understand TGF-βRⅡabnormal in acute and chronic leukemia patients ,our group amplified TGF-βRⅡcoding sequence by PCR in acute leukemia primary cells, and connected to the T vector,then transferred in competent bacteria, Single colonies were picked, and delivered to company, TGF-βRⅡisomers, named TGF-βRIIA and TGF-βRIIB (75bp, No2 exon deletion),were first found in the world, In the previous study, we only picked a single colony . It has some limitations and does not reflect the two isoforms expression in leukemia cells. TGF-βRIIA and TGF-βRIIB, which deleted in the second exon, So we designed the primer in the first exon and the third exon ,Then detected this two isoforms expression in leukemia cells. Avoid the limitations of previous studies and the complicated sequencing methods. Also the functions of these two isomers were studied in this study.The subjects of the thesis were focused on the following aspects:1.The expression of TGF-βRII in normal cells , acute leukemic chronic leukemic and leukemic celllines were detected by RT-PCR.2.Eukaryotic expression plasmids pEFIRES-N-TGFβRⅡA and pEFIRES-P -TGFβRⅡB were set up by using recombinant DNA technique. Using G418 and puromycin,the stable transferction of K562 with recombinant plasmid expressing TGFβRⅡA and TGFβRⅡB were selected.3.Study the effect of TGF-β1 on the TGF-βRⅡA-K562 and TGF-βRⅡB-K562 cell proliferation differentiation and apotosis.then analyse expression of some different genes and protein associated with apoptosis in this progress by relative quantitive RT-PCR and Western-Blot.The main results are as following:1. TGF-βRII A is over express in normal cell, TGFβRⅡB is over express in normal cell in acute leukemic or chronic leukemic cells, TGFβRⅡA and TGFβRⅡB are both low express in K562 and KAR cellline.2. The eukaryotic expression vector of TGFβRⅡA and TGFβRⅡB(named TGFβRⅡA-k562 cell and TGFβRⅡB -k562 cell) were constructed successfully. The recombinant pEFIRES-N-TGFβRⅡ-A and pEFIRES-P -TGFβRⅡ-B were separately transferred into K562 cell line with lipofectamine.3. Using different doses of TGF-β1 on the TGF-βRⅡA-K562 cell and TGF-βRⅡBK562 cell. Data suggests low dose of TGF-β1 (1 ng/ml) can inhibit the proliferation of TGF-βRⅡA-K562 cell. It can also induce the differenciation and apotosis of TGF-βRⅡA-K562 cell .but the same dose of TGF-β1 can not inhibit the proliferation of TGF-βRⅡB-K562 cell, ether inducing the differenciation and apotosis of TGF-βRⅡB-K562 cell. With increasing of TGFβ1, it can also inhibit the proliferation of TGF-βRⅡB-K562 cell, they has dose and affection relationship. But it has low inhibiter than TGFβRⅡ-A K562 cell.4. RT-PCR and Western-Blot, shows the expressions of Smad2/3 in TGFβRⅡ-A K562 cell increased with low dose of TGFβ1(1 ng/ml),but the expressions of Bcl-2,c-myc decreased. It maybe used for explaining the apotosis of leukemic cell. whereas, Smad2/3,Bcl-2,c-myc are not changed in TGFβRⅡ-A K562 cell using the same dose of TGFβ1.Conclusion:1. TGF-βRII A is over express in normal cell, TGFβRⅡB is over express in normal cell in acute leukemic or chronic leukemic cells, TGFβRⅡA and TGFβRⅡB are both low express in K562 and KAR cellline.2. Low dose of TGF-β1 (1ng/ml) can inhibit the proliferation of TGF-βRⅡA-K562 cell. It can also induce the differenciation and apotosis of TGF-βRⅡA-K562 cell .but the same dose of TGF-β1 can not inhibit the proliferation of TGF-βRⅡB-K562 cell, ether inducing the differenciation and apotosis of TGF-βRⅡB-K562 cell .With increasing of TGFβ1,It can also inhibit the proliferation of TGF-βRⅡB -K562 cell,they has dose and affection relationship.but it has low inhibiter than TGFβRⅡ-A K562 cell.3.The expressions of Smad2/3 in TGFβRⅡ-A K562 cell increased with low dose of TGFβ1(1 ng/ml),but the expressions of Bcl-2,c-myc decreased.It maybe used for explaining the apotosis of leukemic cell. whereas, Smad2/3,Bcl-2,c-myc are not changed in TGFβRⅡ-A K562 cell using the same dose of TGFβ1.
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