293 293 293 1C 293 293 293 293 5 75 Ad5 E1 McMaster University F.L.Graham J.S.Miley 1976 DNA 293 Ad5 293 293 Ca2+ 293 293 293 5-6 20 CelliGen CelliGen PlusTM Bioflo3000 6 1200cm2/g 293 Hela CHO 293 1 98% 7-10 25-50
293 Vero CHO Cytodex? Cytopore Cytoline BIOSTAT?B NBS CelliGen CelliGen PlusTM Bioflo3000 Cell-lift 293 1L spinner 10-15 5-10 106/ml 293 293 293S 293S 293S 5-10 7 5 106/ml 5-10 293
1 2 36.5 0.5 39 39-40 1 40-41 1 41-42 1 43 1 3 95% 5% PH PH 7.2-7.4 PH6.8 PH NaHCO3 NaHCO3 CO2 HEPES PH PH 4 1
2 5-20% 1 2 1 2 3 4 5 3 1 500ml 250ml 100ml 2 1cm 200ml 100ml 50ml 25ml 10ml 10ml 3 30mm 60mm 120mm 4 1ml 10ml 5 50ml 30ml 15ml 10ml 5ml 6 1 2-3
2 3 1-2 1 1 5 2 3 6 g ml ml A 63 1000 200000 B 120 C 200 1000 100 100 100
4 3-5 2 2% NaOH 10-20 1% 30 10-20 3 2% NaOH 5% 30 A B C 1 2.5 2 10 10 18 20 3 70% 1 4 8 8-9
B 1 5ml 5ml PMRI 1640 2 PRMI 1640 10-15ml 10ug/ml 1ml 3 1 1 B B 5ml 4-5ml 2 PRNI 1640 5ml 0.06ug/ml 1640 30-40 3 37 0.075M KCL 10% 1 1 4 2 30 5 3 2 0.2ml 1000rpm8 6 3 2 3ml 30 2000rpm8 7 3 1 3ml 30 2000rpm8 8 60% 2 2000rpm8 9 3m1 3 1 10 80 2 11 G 12-30 16 16-30 B 1 2,5% 75% 20-22 PRMI 1640 199 F10 F12 KCL 2 F-12 85% 15% 100u/ml 4-8 3 A 1 13-14 5ml 2 1000rpm10 0.5ml 3 30mm 1 1-2 30-60 4 2ml 48
5-10 5 0.02-0.04ug/ml 10-12 6 37 0.075MKCL 5ml 37 30 0.5-1ml 3 1 7 5ml 30 3 8 80 2-3 9 B 1 2 3 50ml 100ml 10ml 5-10ml 4 48 5-10 5 A 6 0.025% 0.5-1ml/ 1000-2000rpm10 7 37 KCL 10ml 37 30 0.5-1ml 3 1 8 3 2 30 9 10 PHA PHA Go PHA HPA 1 2.5% 75% M199 KCL 2 RPMI 1640 199 90% 10% PHA 0.1ml 3% 10u/ml 2% 100u/ml 10ml 5ml 3 1 2.5% 75% 0.5-1ml 20 36 72 12 1 2 0.02ug/ml 4 10ml 2000rpm8 5 37 0.075MKCL8ml 37 10-20 6
1-2ml 3 2 2-3 800-1000rpm8-10 7 3 2 10ml 30 1500-2000rpm10 2-3 8 1-2 80 2 9 PHA A 1 10g 75% 37 24-48 2 30ml 4 24 70ml 4 24 8-12 100ml 3 10-50ml 3000-4000rpm30 10ml 4 100ml PHA 2ml G5 B1 20g 75% 10 2 100ml 3 4 24 3000rpm30 10ml 4 1 PRMI M199 DMEM 1640 0.025% KCL 2 85-95% 5-15% 100u/ml 3 1 80-90% 2 0.01-0.03ug/ml 6-10 3 A 90% B 0.025% 0.5-1ml/
3-5ml 2000rpm10 4 37 0.075MKCL8ml 20-30 5 3 2 1-2ml 6 800-1000rpm10 3 2 10ml 30 7 0.5ml 1 2.5% 75% 20-22 50ml KCL 2 1 20-50ml 2 0.02-0.04ug/ml 37 4-6 3 1 PH 2 3 4 1 5 6 7 PH PHA
PHA PH PH PHA
PHA
2000 G 850 350 G G 1 Giemsa 1 3.75gGiemsa 2 Giemsa 500ml 250ml 3 60 24 37 72 4 250ml 2 0.9%
3%tris 0.25% Gremsa 3 1 0.9% 100ml 0.25% 1ml 0.025% 4-5 tris PH6.8 37 2 500ml 3 tris PH Giemea 1ml 3 4 37 1 45 60 1 3-5 5 6 4 37 5-10 10 7 25ml Giemsa 2-3
8 Giemsa tris Gremsa tris 9 Giemsa 80 2 SCE SCE DNA DNA SCE SCE 1 5- BudR, DNA BudR DNA BudR DNA BudR DNA BudR DNA Giemsa BudR DNA 2 BudR BudR 5mg 0.5ml 1N NaOH 5ml 1000ug/ml BudR 3 BudR 2 SSC
20W 4 1 BudR 24 BudR 5-15mg/ml 248 3 0.02ug/ml 3 4 2 SSC 50-60 5cm 30-40 5 PH6.8 2%Giemsa 15 6 G BudR fra 1 1 2 MEM-Fra CT199 2
MEM-Fra 90-95% 5-10% 100u/ml PH 7.5 3 X FraX 1969 1977 Xq27 fra X q X MR fra X q27 FraX 1/2500 X MR 1/2-1/3 MR 1-2%
1 1m1 Hanks 1m1 2m1 ( 2000r min 20min 4 ( 2 1) ( ) Hanks 2000r min 10min RPMI l640 2 10 6 m1 2 T B B T RPMI l640 T B RPMI l640 B ( ) 3 CD4 CD8 1 CD4 T SephDdexC 10 RPMll640 85 CD4 2 CD8 Tris IgG( 10 g m1) 4 PBS CD8 T ( 1 l0 7 m1)3m1 4 2 PBS 10m1 PBS RPMI l640 CD4 4 ( ) 37 C02 1 RPMI 1640 75 60 70 1 HeLa 1
NaHC030 HeLa Hanks 0 25 0 02 EDTA (PS) 5 6 (5m1 1m1) 1 HeLa Hanks 2 0 25 EDTA 4 5ml 1min 3 5 10min 37 4 EDTA Hanks 4 5m1 5 1 2 1 3 6 37 30min 48 3 4 [ ] 1 MEM 63 20 15 (P S)1 5 6 NaHCO3 ph 7 2 7 4 199 70 18 10 (P S) 1 5 6 NaHCO3 ph 7 2 7 4 1640 88 10 (P S) 2 1 5 6 NaHCO3 ph 7 2 7 4 1640 96 2 (P S) 1 5 6 NaHCO3 ph 7 2 7 4 2 (K562) K562 RPMI l640 (5m1 1m1) 1 K562 2 K562 3 37 5 C02 24 48h 4 1 3 1 2
0 1ml D Hanks 0 8m1 0 3 0 1mI( ) 4 4 ( ) 10 2 (3 5) 10 5 m1 (1 3) 10 5 m1 [(2 6) 10 5 ml 37Y 2 3 5 7 3 4 5 7 CPE ( 2 ) 5 7 1 3 ( EDTA EDTA ) 2 37 3 5 (dendritic cell,dc) Th TC T GM-CSF IL-4 IFN- HanKs(CMF-HanKs) GM-CSF(hrGM-CSF) hril-4 hr IFN- 1 RPMI1640 RPMI1640+10% FCS +2mmol/LL- +100IU/ml +100 g/ml +1g/L +1 mmol/l +5 10 5 mol/l 2 2 RPMI1640/IL-4/GM-CSF: RPMI +IL-4(40ng/ml)+GM-CSF(50ng/ml). 3 RPMI1640/IFN- : RPMI + IFN- 1 ~ 10ng/ml 25 ml 75 ml CO2 3
1 2 IL-4 GM-CSF RPMI1640 1 10 6 /ml 5% CO2 37 3 3 3 IL-4 GM-CSF 1640 CO2 4 IL-4 GM-CSF 7 IL-4 GM-CSF 1640 IFN- RPMI1640 5%CO2 37 9 1 90% 2 CD14 90% ~ 100% IL-4 GM-CSF 7 IFN- 9 CD14 MHC- CD1 3 4 CD34 + CD34 + 5 LAK LAK(Lymphokine activated killer cell) IL-2 IL-12 3~5 LAK IL-2 hr12 LAK LAK MHC. CMF-Hanks. 1 RPMI1640 (RPMI+10mmol/LHEPES+2mmol/L + 250 g/ml ) 2 LAK RPMI1640 +10% AB +IL-2 1000 /ml. 3 X-VIVO 15. 6 75cm2 CO2 1 (PBMC) O 40ml 3 IL-2 CMF-Hanks 2000r/min 20min PBMC 2 CMF-Hanks PBSPBMC2 LAK RPMI1640 +10%AB +1000 /ml IL-2 1000 /ml IL-2 X-VIVO 15 PBMC 2x10 6 /ml 4
6 5%CO2 37 4 ~ 5 3 280Kg 5min -20 TIL 4 RPMI1640 X-VIVO15 2 5 5x10 8 ~ 1x10 9 FACS CTL NK [ ] 1 LAK NK CD56 + CD3 - CD25 + T CTL CD3 + CD8 + CTL CD56 + NK RaJi 2 LAK AIM-V 10% RPMI1640 X-VIVO15 LAK TIL tumor infiltrating lymphocyte,til IL 2 TIL LAK IL 2 100 1640 75 RPMI1640 10 AB IL 2 200u/ml X VIVO15 IV 230u/g I DNA 3600u V 1500u/g 75cm 2 15ml CO2 1 RPMI1640 1 2mm 3 3600u DNA 50µg 125u RPMI1640 40ml 37 1 2 2 200 1500r/min RPMI1640 X VIVO15 2 3 TIL 2 2 100 75 2000r/min 20min 100 TIL 75 RPMI1640 X VIVO15 TIL 2 4 2 TIL 10 AB 20 LAK 200u/ml IL 2 RPMI1640 20 LAK 200u/ml IL 2 X VIVO15 RPMI1640 3 5 10 5 ml, 75cm 2 5 CO2 37 1 2 15 20 TIL 3 5
1 TIL 50u/ml 1000u/ml IL 2 LAK 1000u/ml IL 2 2 TIL 70 3 feeder cell, EB B B EBV TIL TIL TIL T T NK T T 1 IL 2 PHA PBMC T T T 50GY PBMC RPMI1640 RPMI1640 1mmol/L 2mmol/L 100u/ml 100 g/ml 10 AB PHA A 10 4 u/ml hril 2 PBS 1 BSA 96 U 24 CO2 1 1 0 5 1 5 96 U 10 96 2 5 10 5 ml RPMI1640 20u/ml IL 2 1µg/ml PHA A 3 3 96 100µl 50000 4 T 6
20u/ml IL 2 T 10 6 ml 10 5 10 3 ml C 2 3 5 2 3 3 15ml 5ml CM 20u/ml IL 2 4 96 1 1 50µl C 50 96 50µl/ 0 5 2 2 100µl C 100 96 50µl/ 1 3 3 4 500µl C 500 3 500 5mlCM, 96 50µl/ 5 ; 4 500 2 5mlCM, 50µl/ 10 4 96 50µl T 100µl IL 2 20u/ml 2 T 1 2 4 2 3 50µl 50µl IL 2 20u/ml 10 15 50µl 50µl 50000 IL 2 20u/ml PHA A 1µg/ml 2 21 25 10 1 5 10 3 0 5 1 100µl 96 2 50µl/ 50000 4 4 8 8 24 1 5 T 2 3 IL 2 20u/ml 10 15 IL 2 20u/ml PHA 1µg/ml T 0 5 1 10 6 10 6 1 TCRV RT PCR TCR V 2 3 T PHA 4 T HLA NK 7
NK NK NK NK PHA 100µg/ml IL 2 ril 2 10 5 u/ml 0 5ml/ 20 4 IL 2 1 RPMI1640 10 FCS 0 5µg/ml PHA 200u/ml IL 2 10g/L 100u/ml 100µg/ml 1mmol/L 2 RPMI1640 10 FCS 10g/L 100u/ml 100µg/ml 1mmol/L 3 NK RPMI1640 10 FCS 10g/L 100u/ml 100µg/ml 1mmol/L 0 2u 100u/ml IL 2 4 RPMI1640 10 FCS PBMC 96 CO2 1 NK 1 NK 2 NK 10 6 ml 2 NK 1 PBMC 2 4 300 g 12min 3 2 10 6 ml 4 5000rad 4 300 g 12min 2 10 6 ml 5 6 96 100µl 60ml 1 2 10 8 6 96 37 NK 7 NK 96 100µlNK 10 5 2 5 8 5 CO2 37 3 NK 2 3 1 100µl 2 2 10 6 ml NK 100µl 1 50ml 2 30ml 5000rad 3 300 g 12min 4 NK 2 10 6 ml 4 6 8 100µl NK 100µl 5 NK 9 10 8 6 11 15 1 96 NK 1 2 2 8
4 4 8 2 96 7 1 2 NK 96 NK 96 3 NK 1 PCR PCR 16srRNA PCR 16sr RNA EB 16sr DNA 40umol/L 1. 5 -ACTCCTACGGGAGGCAGCAGTA-3 2. 5 -TGCACCATCTGTCACTCTGTTAACCTC-3 pbr322 40umol/L. 1. 5 -CATCTCGGGCAGCGTTGGGT-3 2. 5 -AGCGCAAGCGGAGTCAGTGAGC-3 10mmol/L Tris-HCL(pH8.3), 50mmol/L KCL, 2.5mmol/L Mgcl 2, 5g/L Tween20 5g/L TritonX-100 K proteinase K K 20 mg/ml 50% -20 0 C DNA Taq 10X TAE A1 10mg/ml 6X 40% 0.125% 125mmol/L EDTA. DNA 50ng/ml pbr322 DNA DNA UV PCR 0.5ml 0.2ml 1) 9
1 10 6 1.5ml 15min 2 100ul K 60ug/ml 3 60 0 C 1h 95 0 C 10min 2) PCR 1 PCR 10X PCR 25mmol/L dntps 40umol/L 16sr DNA 40umol/L pbr322 DNA pbr322 DNA 1pg/ml DNA 5U/ml 5.0ml 0.4ul 1.0ul 2.0ul 2.0ul 0.2ul 35.4ul 45ul 2 1:10 1:100 3 eppendorf 45ul PCR 1:10 1:100 5ul 4 eppendorf 50ul DNA 5 DNA 95 0 C 10min 95 0 C 30S 58 0 C 1min 72 0 C 1min, 30 72 0 C 10min 3 PCR 1 EB 0.1ug/ml 1% TAE 2 PCR 10ulPCR 2ul 10ul DNA 3 TAE 80V 60-90min 4 EB DNA DNA 1 1 DNA 10ngDNA 2-20 0 C 2 1 45ul PCR 5ul 2 45ul PCR eppendorf 2ul pbr322 DNA 1pg/ml 3 PCR pbr322 10 5 2-3 DNA Taq 2 DNA DNA Hoechst 4-6- -2 4-6-diamidino-2-phenylindole DAPI DNA 10
CKI 1 IFO5003 JCRB0008 Vero ATCC CCL82 10 6 1ml mycoplasma hyorhinis (ATCC 29052) M.orale(IFO 14477) MEM 10 FBS PBS 3 1 100mlPBS Hoechst Dye 5mg thimerosal 10mg 30min 1ml 20 0 15ml 100mlPBS 0.075 g/ml Hoechst Dye 0 15 g/ml thomerosal 30min 60mm 10ml 4 1 3 3 2 3 4 1200r/min, 10min 6 1ml 20 30min 37 2 4 1200r/min 10min, 2 196 CKI 1 Vero 37 1 2 3 10 3 ml 4 1ml CO2 24h 160 240 0 9ml i 0.1ml ii 0.1ml iii iv 100cfu/0.1ml M.hyorhinis M.orale 4 CO2 6 3 CMF PBS 1ml 10min ml 30min, 3 400 600 4 1000 5 1 CKI 1 vero 3T6 ARCCCCL96 2 3 Hoechst DAPI 11
4, 80 5 M.hyorhinis M.orale 6 DNA DNA M.hyorhinis M.orale M.salivarium M.hominis M.fermentans 97 1cfu 10 3 10 8 cfu/ml DNA RNA / BM-cyclin 25cm 2 CO 2 1. 1 10 5 /ml 25cm 2 10ml IC 50 5~10 2 48h 1 3 1 4 2 2 3 2. 3. 96 4. 41 0 C 5~10h 18h 41 0 C 50 0 C 4~6min 37 0 C 1~3 limulus amoebocyte lysate,lal LAL 12
LAL LAL 0.125EU ml ( ) CSE LAL 10mm, 75mm ( ) 0 1mol/L 0 1 mol/l 37 1 200 1000 l 10 5 2 1ml PH PH 250 1 parafilm 1 LAL 2 1 1 CSE LAL CSE 2 1 0.5,0.25 0.125EU ml 0.25,0.125,0.0625 0.0312EU/ml ( 0.1ml) 2 0.1mlCSE 0.1ml LAL 3 4 37 1 60 180 CSE 5 GEP 10 m,m= e/f, e log f GEP 0.5 2 2 1 CSE 2 ph NaOH HCL ph 6 5 7 5 3 GEP GEP GEP 0 5 2 3 1 A 2 0 5 CSE (0 1ml LAL ) B LAL ( 0 1m1 ) C LAL (0 1m1) D CSE CSE 2 13
2 (0 1m1) 0 1m1 LAL 3 ( ) ( ) 4 B 1 CSE RSE CSE BSE 2 CSE 3 4 250 30min 1 1 1000 1000EU/ml CSE 2 1ml CSE 3EU CSE 3 60 30min 250 30min 4 LAL 5min LAL 5 3 log 2 3 log 3 log 1000 1EU 10000 10EU LAL 25030min LAL 60,30min 250 30min 1 250 15 2 1000EU 10000EU 3 14
4 3 log 1. Immunological techniques made easy P24-34.P68-72. 2. Rosenberg,S A. Lymphokine-activated killer cells: a new approach to the immunotherapy of cancer. JNCI 1985,75:595-603. 3. Rosenberg,S A. a new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 1986.223:1318-1321 4. 1999 5. P62 65. 15