B99017024 00 0

-------------------------------------------------------------------------------------------------- 1 -------------------------------------------------------------------------------------------------- 2 ---------------------------------------------------------------------------------------------------------- 4 "Cells-followed-by-CP" 7 ------------------------------------------------------------------------------------------ 8 ---------------------------------------------------------------------------------------------------- 15 ---------------------------------------------------------------------------------------------------- 26 CTLA4Ig 31 ------------------------------------------------------------------------------------------ 32 ---------------------------------------------------------------------------------------------------- 49 ---------------------------------------------------------------------------------------------------- 57 ---------------------------------------------------------------------------------------------------------- 64 -------------------------------------------------------------------------------------------------- 65 ---------------------------------------------------------------------------------- 70 -------------------------------------------------------------------------------------------------- 73 --------------------------------------------------- 73 ---------------------------- 82 -------------------------------------------------------------------------------------------------- 98 ------------------------------------------------------------------------------------------ 98 ---------------------------------------------------------------------------------------------------------- 100 1

Cells-followed-by-CP CFCP CTLA4Ig,, 1 CFCP, LEW DA 2 (CP) 4, 2 DA LEW 2 DA LEW, 2 CP 4 3 CTLA4Ig, CTLA4Ig 4 4 RT-PCR CTLA4Ig MLR CTL Th1/Th2 CFCP CTLA4Ig, MLR CTL IL-2 MLR Th1/Th2 CTLA4Ig, CTLA4Ig, CTLA4Ig CFCP CTLA4Ig T (anergy) CTLA4Ig CTLA4Ig T, CTLA4Ig 2

Study on the Induction of cardiac transplantation tolerance in rats by establishing allogeneic mixed chimerism ABSTRACT Objective To induce allogeneic cardiac transplantation tolerance in recipient rats by Cells-followed-by-CP system or administration of adenovirus containing CTLA4Ig gene, to obtain a robust tolerance and improve the survival of allograft, a chimerism has been established by infusing allogeneic bone morrow cells into the donor rats treated by above regimens. Methods The cardiac transplantation tolerance was induced according to Cells-followed-by-CP system before transplantation, i.e., the recipient LEW rats were infused with spleen cells (SC) though portal venous (p.v.) on day 0, and then were administrated intraperitoneally(i.p.) with cyclophosphamide (CP) on day 2, were infused with bone marrow cells (BMC) of DA rats by glossal vein (g.v.) on day 4. After 2 weeks heart grafting was carried out In perioperative and postoperative regimen of tolerance induction, the donor spleen cells were infused p.v. during the same time of operation from DA to LEW rats on day 0, the cyclophosphamide (CP) was injected i.p. on day 2, and the BMC were infused by glossal vein (g.v.) on day 4. The adenoviral vectors containing CTLA4Ig gene were constructed and used in induction of tolerance. During cardiac transplantation, the adenoviral vectors were injected g.v. and the SC were infused p.v. immediately after grafting, the BMC were infused g.v. on day 4. The survival time was observed and monitored. The expression of CTLA4Ig gene was determined by RT-PCR in vivo. The tolerant mechanism was investigated by using adoptive transfer tests, MLR and its reverse by IL-2, detection of CTL activity, determination of Th1/Th2 type cytokines, analysis of chimerism. Results The cardiac transplantation tolerance can be induced by Cells-followed-by-CP system or administration of adenovirus containing CTLA4Ig. On the basis of above treatment, infusion of BMC can significantly prolong cardiac allograft survival further, and prolong skin allograft survival; This tolerance can be transferred adoptively; The MLR and CTL activity were decreased donor-specifically, and the decreased MLR were reversed by exogenous IL-2 in vitro; The degree of chimerism was enhanced remarkably. The polarization of Th1 and Th2 type cytokines are not observed. There are different 3

expressions of CTLA4Ig gene in different tissue, and the expression of CTLA4Ig gradually decreases depending on time in vivo. Conclusions Infusion of BMC can increase the degree of chimerism and augment the tolerance, which is induced by Cells-followed-by-CP system or administration of adenovirus containing CTLA4Ig gene. This transplantation tolerance involved multi-mechanisms, such as mixed chimerism, regulatory T cells, and anergy. The adenoviral vectors containing CTLA4Ig gene were constructed successfully. Expressed CTLA4Ig protein can effectively inhibit T cells activity and induce transplantation tolerance in recipients when the rats were administrated with the adenoviral vectors containing CTLA4Ig. Periopertive and postoperative induction of transplantation tolerance is an effective, and maybe clinically practical. Keyword: Transplantation tolerance; Bone marrow cells; Mixed chimerism; Cardiac transplantation; CTLA4Ig gene therapy 4

,, ( ) (Mixed chimerism) (1) T ( ) (2) T ( ) (Total body irradiation, TBI) (1), T,, (2,3) T (Donor-specific T cells) (4,5) (Bone marrow transplantation, BMT) (6),, (GVHD) (7) 5

(8) CD40L CTLA4Ig (9 10) Durham (9) 8 90 Cells-followed-by-CP, Anti-Ia Anti-TCR Cells-followed-by-CP,,, CTLA4Ig T T T, Cells-followed-by-CP 6

, CTLA4Ig, CTLA4Ig, 7

"Cells-followed-by-CP" 1960, ( ), (Cells-Followed-by-CP System) Cells-Followed-by-CP System 1 10 8 ( ) 2 (1-3 ) 150-200mg/kg, Cells-Followed-by-CP 8

( ) 1 GX-SS.22-3) JZ9708 2 DA(RTl a ) LEW(RT1 l ) SD 8-16 150-250g 3 (HCA ) 75% 5% 10% FITC RT-1a Pharmingen 2 (IL 2) 4 4.1 Hanks 4.1.1 : NaCl 160g KCl 8.0g,MgSO4.7H2O 2g MgCl.6H2O 2g 9

800ml ; CaCl2 2.8g, 100ml ;, 1000ml, 2ml 4.1.2 Na2HPO4.12H2O 3.04g, KH2PO41.2g, 20g, 800ml ; 0.4% 100ml,, 1000 ml, 2ml 4.1.3 1 1 18 8 (0.07Mpa),20, 4,, 3.5%NaHCO3 ph 7.2-7.4 4.2 RPMI-1640 RPMI-1640 10.4g, Hepes 5.95g, L- 300mg,NaHCO 3 2.1g, 0.11g, 5mM 2-ME 2ml, G 10, 10, 1000ml,, 3.5%NaHCO 3 ph 7.2 1640 (IC-1640) 10% FCS 1640 (C-1640) 4 4.3 G 100 1.0g, 10ml, 20ml, 5, 50mg, -20 4.4 Tris 206mg NH 4 Cl 747mg 100ml 1N HCl ph 7.2, 4 4.5 3ml 97ml 3%(v/v) 4 4.6 (PBS ph7.4,0.01m) NaH 2 PO 4.2H 2 O 0.296g, Na 2 HPO 4.12H 2 O 2.9035g,NaCl 8.19g, 1000ml, 4 4.7 (PBS PH7.2,0.15M):NaCl 8.0g,KCl 0.20g, Na 2 HPO 4 1.15g, KH 2 PO 4 0.20g, 1000ml, 4 4.8 2% 1%HCHO 0.1%NaN 3 PBS(pH7.4),, 4 4.9 FACS 1% BSA 0.1% NaN3 PBS(0.15M,pH7.2), 4 10

( ) (11) 1 1-1 AO: PA: LA: RA: LV: RV: 1-1. Fig1-1. Schematic illustration of heterotopic rats cardiac transplantation. 1.1 6 1% 50mg/kg 70% 1.2 1.2.1 DA 10ml(10%, 200 11

) 3-4 1.2.2 7 9-0 7 0-4 1.3 (7 ) 0.5cm 1.4 1.4.1 9-0 1 2 1 2 2 1 1.4.2 1 12

1.5, 24 2 2 LEW DA DA SD 2cm,, LEW 8 1 3 1% (30-50mg/kg ) Hanks 100 1000rpm 10min 2min Hanks 1000rpm 10min RPMI1640 0.2% 4 10ml Hanks 200g 10min 100 1500rpm 10min Hanks 0.2% 5 Cells-Followed-by-CP System LEW (Bone marrow cells, BMC), 13

LEW 5 5.1 5.2 (80mg/kg ) 80mg/kg LEW 5.3 LEW 3 10 8 5.4 2d (80mg/kg) 5.5 4d, (3 10 8 ) 15d DA LEW 6 Cells-Followed-by-CP System ( ) 6.1 (SC/CP) DA LEW LEW 3 10 8 DA (80mg/kg ) 6.2 (SC/CP/BMC) 4 LEW 3 10 8 DA 6.3 DA LEW 6.4 LEW 80mg/kg 6.5 LEW DA (3 10 8 ) 7 (WBC) LEW 14

1.5 10 6 /ml, 1ml FITC RT-1 a 2µl( 1µg), 37 40min PBS (ph7.4) 3 FACScan-440 8 (MLR) LEW (5 10 6 /ml) DA SD 2500rad 100 l 96 3 37 C 5%CO 2 4 3 H-TdR 1µCi/ 20-24, CPM 9 9.1 (DTH), 15 LEW 3 10 8 /ml (1ml), LEW DA DTH 9.2 30d LEW 2 10 8 /ml LEW ( 450rad ) 24h (12), DA 10 IL 2 LEW MLR DA, LEW, LEW 20µl (1 IU/µl) IL 2, MLR IL 2 11 T (CTL) 50d LEW (3 10 7 ) DA 15