Nuôi cấy hoạt hóa và đánh giá tính đáp ứng miễn dịch của tế bào lympho tách từ bệnh nhân ung thư phổi

1 B GIO DC V O TO TRNG I HC Y H NI *** B Y T O MNH PHNG NuôI cấy hoạt hóa đánh giá tính đáp ứng miễn dịch tế bào lympho tách từ bệnh nhân ung th phổi LUN VN BC S NI TR H NI - 2016B GIO DC V O TO B Y T TRNG I HC Y H NI *** O MNH PHNG NuôI cấy hoạt hóa đánh giá tính đáp ứng miễn dịch tế bào lympho tách từ bệnh nhân ung th phổi Chuyờn ngnh : Húa sinh y hc LUN VN BC S NI TR NGI HNG DN KHOA HC TS Trn Huy Thnh H NI 2016 LI CM N Vi lũng kớnh trng v bit n sõu sc, tụi xin gi li cm n chõn thnh ti: TS BS Trn Huy Thnh, phú trng b mụn Hoỏ sinh trng i hc Y H Ni, ngi hng dn khoa hc, ngi thy ó trc tip ch bo, tn tỡnh truyn t nhng kin thc, kinh nghim quý bỏu v to mi iu kin thun li sut quỏ trỡnh hc v nghiờn cu tụi cú th hon thnh lun ny GS TS BS T Thnh Vn, phú hiu trng trng i hc Y H Ni, trng b mụn Hoỏ sinh, gim c Trung tõm nghiờn cu gen- protein trng i hc Y H Ni, ch nhim ti cp b Tip nhn v nghiờn cu phỏt trin cụng ngh nn t bo dch iu tr ung th t Nht Bn v Vit Nam ó giỳp v to mi iu kin thun li cho tụi sut quỏ trỡnh hc cng nh cho phộp tụi tham gia thc hin ti ny TS BS Nguyn Thanh Bỡnh, ging viờn b mụn Sinh lý bnh- Min dch trng i hc Y H Ni, ngi thy ó tn tỡnh giỳp , ch bo, chia s nhng kinh nghim quý bỏu hc v nghiờn cu PGS TS Phm Thin Ngc, nguyờn trng b mụn Hoỏ sinh trng i hc Y H Ni, ó giỳp , to iu kin cho tụi sut quỏ trỡnh hc ti b mụn Tụi xin gi li cm n sõu sc ti cỏc thy cụ, cỏn b ging dy, k thut viờn v y cụng b mụn Hoỏ sinh trng i hc Y H Ni, ó ch bo, giỳp tụi sut quỏ trỡnh hc ti b mụn Tụi xin gi li cm n chõn thnh ti TS Trn Võn Khỏnh cựng ton th cỏn b Trung tõm nghiờn cu gen- protein trng i hc Y H Ni ó tn tỡnh giỳp tụi quỏ trỡnh hc v nghiờn cu Tụi xin by t lũng cm n sõu sc ti TS Nguyn Vn ụ, phú trng b mụn Sinh lý bnh- Min dch trng i hc Y H Ni ó to iu kin giỳp , cho phộp tụi hc cỏc k thut v chia s cỏc kinh nghim quý bỏu Tụi xin gi li cm n chõn thnh n Ban Giỏm hiu, phũng o to Sau i hc trng i hc Y H Ni ó to iu kin cho tụi c hc v nghiờn cu Tụi xin gi li cm n sõu sc n cỏc bnh nhõn v ngi tỡnh nguyn ó giỳp tụi cú th thc hin c nghiờn cu ny Cui cựng tụi xin ghi nh cụng n sinh thnh, nuụi dng v tỡnh yờu thng ca cha m tụi, cng nh s giỳp ca cỏc anh em gia ỡnh, nhng ngi ó luụn bờn tụi, l ch da vng chc cho tụi yờn tõm hc v hon thnh lun H Ni, ngy 09 thỏng 12 nm 2016 Bỏc s ni trỳ o Mnh Phng LI CAM OAN Tụi l o Mnh Phng, bỏc s ni trỳ khoỏ 38, chuyờn ngnh Hoỏ sinh y hc Tụi xin cam oan õy l cụng trỡnh nghiờn cu ca tụi, chớnh tụi thc hin di s hng dn ca TS Trn Huy Thnh.Cỏc s liu, kt qu nghiờn cu nờu lun l trung thc v cha tng c cụng b bt k nghiờn cu no khỏc H ni, ngy 09 thỏng 12 nm 2016 Ngi cam oan o Mnh Phng CC CH VIT TT AIET (Autologous Immune Enhancerment Therapy) DC (Dendritic cells) DNA ELISA (Enzyme Linked Immunosorbent Assay) FDA (U.S Food and Drug Administration) IL-2 NSCLC (non-small cell lung cancer) PCR (Polymerase Chain Reaction) RNA SCLC (Small Cell Lung Cancer) TAAs (Tumor Associated Antigens) TNF (Tumor Necrosis Factor) UTP Liu phỏp truyn t bo dch t thõn T bo tua Deoxyribonucleic acid Xột nghim mim dch gn men Cc qun lý thc phm v dc phm Hoa K Interleukin Ung th phi khụng phi t bo nh Phn ng tng hp chui Ribonucleic acid Ung th phi t bo nh Khỏng nguyờn liờn kt u Yu t hoi t u Ung th phi MC LC DANH MC BNG 10 T VN ấ Ung th phi (UTP) l bnh cú t l mc v t vong hng u s cỏc loi ung th trờn th gii Theo cụng b ca c quan nghiờn cu ung th quc t (International Agency for Research on Cancer: IARC), t chc y t th gii (World Health Orgnization: WHO) vo nm 2012, trờn ton th gii s bnh nhõn ung th phi mi mc l khong 1.8 triu v s bnh nhõn t vong ung th phi lờn ti 1.59 triu[1] Ti Hoa K, mt nc cú nn y t phỏt trin, c tớnh nm 2016 s cú ti 224390 bnh nhõn mi mc v 158080 trng hp t vong ung th phi[2] Ti Vit Nam, nhng ghi nhn v tỡnh hỡnh ung th trờn c nc cng cho thy, UTP l mt nhng loi ung th ng hng u nam gii v cng l mt cỏc loi ung th hng u n gii[3], [4] Mt iu ỏng bỏo ng cho cỏc quc gia ang phỏt trin nh Vit Nam l ti cỏc nc phỏt trin, t l mc UTP ó cú xu hng gim xung thỡ ti cỏc nc ang phỏt trin, t l mc v t vong UTP ngy mt tng cao [5] V iu tr UTP, phng phỏp iu tr ph thuc vo týp mụ bnh hc (ung th phi khụng phi t bo nh- NSCLC hay ung th phi t bo nhSCLC) v cỏc c im khỏc ca ung th nh giai on bnh[2] Cỏc la chn iu tr bao gm phu thut, hoỏ tr, x tr v liu phỏp iu tr trỳng ớch Tuy nhiờn, ch 16% bnh nhõn ung th phi c chn oỏn giai on ung th cũn khu trỳ [2] Vi cỏc bnh nhõn chn oỏn giai on mun, khụng cũn kh nng phu thut, ú cỏc phng phỏp húa tr, x tr c ch nh nhng hiu qu hn ch v thng mang li nhiu tỏc dng khụng mong mun[6] Nhng nm gn õy cỏc phng phỏp iu tr trỳng ớch i em n nhng bc tin mi iu tr UTP c bit l ung th phi khụng phi t bo nh (Non- Small Cell Lung Cancer: NSCLC)[7], [8] 51 CHNG BN LUN 4.1 p dng quy trỡnh tỏch chit v nuụi cy hot húa t bo lympho t mỏu ngoi vi 4.1.1 Quy trỡnh tỏch chit t bo lympho t mỏu ngoi vi - Trong s ngi tỡnh nguyn kho mnh, cú nam (chim t l 4/5) v n (chim t l 1/5) Tui trung bỡnh ca nhúm ngi tỡnh nguyn l 29.6 nghiờn cu ny, i tng ngi tỡnh nguyn c chn l nhng ngi kho mnh v vi mc ớch th nghim, xõy dng cỏc quy trỡnh nghiờn cu nờn khụng bt buc tng ng vi nhúm bnh nhõn v cỏc yu t nh tui, t l gii tớnh - S lng t bo bch cu tỏch c t 10ml mỏu ngoi vi ngi tỡnh nguyn kho mnh trung bỡnh l 6.23 ì 106, thp nht l 2.32 ì 106, cao nht l 8.595 ì 106 Trong cỏc mu ny,2 mu MS01 v MS02 c tỏch cựng thi im S lng t bo thu c sau tỏch cú xu hng tng dn cho thy s tin b k thut tỏch t bo - Trong qun th t bo lympho T, lympho B v NK thu c sau tỏch t mỏu tnh mch ngoi vi ngi tỡnh nguyn kho mnh cú t l t bo lympho T (CD3+) trung bỡnh chim ti 78.256% trung bỡnh l 7.812% iu ny cho thy k thut tỏch t bo lympho mỏu ngoi vi bng phng phỏp ly tõm thay i t trng s dng Ficoll ỏng tin cy Nh vy k thut tỏch t bo lympho t mỏu ngoi vi th phi ó c ỏp dng thnh cụng trờn i tng l ngi tỡnh nguyn kho mnh - S lng t bo trung bỡnh thu c sau nuụi cy t bo tỏch t mỏu tnh mch ngoi vi ngi tỡnh nguyn l 4.057 ì 109 Trong s cỏc mu ny 52 mu MS03 ó t mc tiờu ca quỏ trỡnh nuụi cy (ớt nht 5ì109) t bo Kt qu ca mu MS04 v MS05 gim thp hn so vi cỏc mu khỏc hai mu nghiờn cu ny, nhúm nghiờn cu ó th nghim thay i thi gian cy chuyn Tuy nhiờn kt qu cho thy s thay i ny khụng em li hiu qu mong mun 4.1.2 Quy trỡnh nuụi cy t bo lympho thu c sau tỏch t mỏu ngoi vi - Trong mu t bo thu c sau nuụi cy t bo tỏch t mỏu tnh mch ngoi vi ngi tỡnh nguyn kho mnh, cú mu ỏnh giỏ c t l cỏc loi t bo C mu cũn nh cha ỏnh giỏ ý ngha thng kờ nhiờn nhúm nghiờn cu cng ghi nhn s tng t l t bo lympho T (CD3+) s cỏc t bo lympho T, lympho B v NKso vi thi im trc nuụi cy t 78.256 6.864 % lờn 98.625 0.624% T l t bo lympho TCD8+, t bo hiu lc chớnh liu phỏp iu tr c nghiờn cu ny hng ti cng cú xu hng tng, t 29.316 5.606 % lờn 62.12 22.494 % Tuy nhiờn mu MS05 t l ny gim t 37.89% xung 31.79% S liu ny cng gi ý vic iu chnh thi im cy chuyn ó tin hnh khụng mang li hiu qu mong mun - T l t bo lympho B (CD19+) v t bo NK (CD16+ CD56+) sau nuụi cy gim so vi trc nuụi cy (ln lt t 11.40 8.176 v 7.8121.811% gim cũn 1.04 0.472 % v 0% Tuy nhiờn v s lng tuyt i ca t bo NK li tng so vi trc nuụi cy - cỏc mu t bo nuụi cy khụng thy tng s biu hin mRNA IL-2 iu ny cú th liờ quan n quỏ trỡnh tỏch chit RNA t t bo lympho v bo qun RNA hoc quỏ trỡnh hot hoỏ cỏc t bo lympho õy l iu nhúm nghiờn cu s phi tip tc tỡm hiu v iu chnh cú th t c nhng kt qu tt hn 53 - Trong cỏc mu t bo nuụi cy ch cú mu MS04 s biu hin mRNA ca cỏc t bo thu c sau nuụi cy tng biu hin gp 7.06 ln so vi trc nuụi cy Tng t vi s biu hin mRNA IL-2, õy cng l iu nhúm nghiờn cu s phi iu chnh cú th t c nhng kt qu tt hn - mu c ỏnh giỏ (MS04 v MS05) u khụng phỏt hin s nhim mycoplasma dung dch t bo thu c sau nuụi cy iu ny gi ý rng quỏ trỡnh nuụi cy t bo m bo v sn phm t bo thu c l an ton - mu c ỏnh giỏ (MS04 v MS05) u khụng phỏt hin s nhim vi khun dung dch t bo thu c sau nuụi cy iu ny cng l mt bng chng gi ý rng quỏ trỡnh nuụi cy t bo m bo v sn phm t bo thu c l an ton - Hai bnh nhõn ung th phi u l ung th phi khụng phi t bo nh l týp mụ bnh hc thng gp hn C hai bnh nhõn u giai on III phự hp vi tiờu chun la chn ó t - S lng t bo bch cu trung bỡnh thu c t hai bnh nhõn ung th phi l 1.45 ì 106 Mc dự c mu cũn nh cha ỏnh giỏ mc ý ngha thng kờ xong cỏc s liu cng cho thy s lng t bo tỏch c t 10ml mỏu tnh mch ngoi vi bnh nhõn ung th thp hn hn s liu thu c t 10ml mỏu tnh mch ngoi vi t ngi tỡnh nguyn kho mnh Ngoi so sỏnh vi s liu cựng i tng l cỏc bnh nhõn ung th nghiờn cu khỏc, kt qu nghiờn cu ny l tng t vi cụng b ca Kazuro Iwai v cng s nm 2012 vi kt qu >1 ì 106 t bo (over ì 106 harvested cells) tin hnh trờn 540 bnh nhõn mc cỏc loi ung th khỏc (bao gm c ung th phi) 54 - Trong s bnh nhõn ung th phi õy, bnh nhõn BN02 mi kt thỳc hoỏ tr t cỏch ngy ú s lng t bo bch cu tỏch c rt thp, ch 0.50 ì 106 t bo iu ny cng phự hp vi s suy gim chc nng tu xng m hu qu l gim bch cu mỏu ngoi vi bnh nhõn ung th iu tr hoỏ cht núi chung Kt qu ny cng gi ý vic i thi gian bnh nhõn hi phc sau iu tr v ỏnh giỏ s lng bch cu mỏu ngoi vi nhng bnh nhõn ny trc tin hnh ly mỏu tỏch t bo lympho - Qun th t bo lympho T, lympho B v NK thu c cú t l t bo lympho T (CD3+) trung bỡnh l 82.39%, tng t nh kt qu thu c nhúm ngi tỡnh nguyn kho mnh Nh vy k thut tỏch t bo lympho t mỏu tnh mch ngoi vi t kt qu mong mun vi mu BN1 - S lng t bo lympho thu c sau nuụi cy mu BN1 l 5.796 ì 109, ó t mc tiờu Kt qu ny cng tng t vi s liu c cụng b nghiờn cu ca Karuzo Iwai nm 2012 (5ì 109) hay cụng b caSchuster SJ v cng s vi 6.81ì 109 t bo S lng t bo thu c sau nuụi cy mu BN2 ch t 1.904 ì 109 iu ny mt ln na cho thy cn i thi gian bnh nhõn hi phc sau iu tr v ỏnh giỏ s lng bch cu mỏu ngoi vi nhng bnh nhõn ny trc tin hnh ly mỏu tỏch t bo lympho 4.2.Bc u ỏnh giỏ tớnh ỏp ng dch ca t bo lympho tỏch t bnh nhõn ung th phi sau nuụi cy hot húa - T l t bo lympho T (CD3+) trung bỡnh qun th t bo lympho T, lympho B v NK thu c sau nuụi cy l 95.27% chng t quỏ trỡnh nuụi cy t bo t hiu qu, tng sinh cỏc t bo lympho T 55 - T l t bo lympho TCD8 + trung bỡnh v lymho TCD4+ trung bỡnhln lt l 52.06 4.497% v 34.83 1.287% T l ny cú s khỏc bit vi cụng b ca Karuzo v cng s nm 2012 vi cỏc s liu ln lt l 6115% v 30 15% Tuy nhiờn ý ngha thng kờ ca s khỏc bit l cha th xỏc nh - cỏc mu t bo thu c sau nuụi cy khụng thy tng s biu hin mRNA IL-2 v mRNA TNF so vi thi im trc nuụi cy tng t cỏc mu ngi tỡnh nguyn kho mnh õy l nhúm nghiờn cu cn tip tc tỡm hiu v iu chnh - Vi c mu BN01 v BN02 u khụng phỏt hin s nhim mycoplasma hay vi khun dung dch t bo thu c sau nuụi cy iu ny gi ý rng quỏ trỡnh nuụi cy t bo m bo v sn phm t bo thu c l an ton 56 KT LUN 5.1.p dng quy trỡnh tỏch chit v nuụi cy hot húa t bo lympho t mỏu ngoi vi: - T mu 10ml mỏu tnh mch ngoi vi ca ngi tỡnh nguyn kho mnh ó tỏch c trung bỡnh l 6.23 ì 106 2.446ì 106t bo Trong s cỏc t bo lympho T, lympho B v NK trung bỡnh thu c 78.256% t bo lympho T, vi t l t bo TCD8 trung bỡnh l 29.316% - Sau nuụi cỏc t bo tỏch t mỏu tnh mch ngoi vi ngi tỡnh nguyn, s lng t bo trung bỡnh thu l 4.057 ì 109 ú kt qu mu MS03 l 8.908 ì 109 t mc tiờu S thay i thi im cy chuyn mu MS04 v MS05 khụng t hiu qu mong mun - T l t bo lympho trung bỡnh qun th t bo lympho t, lympho B v NK thu c sau nuụi cy l 98.625%, ú t l t bo TCD8 + trung bỡnh l 62.12% - Khụng phỏt hin s tng biu hin mRNA IL-2 v TNF cỏc t bo thu c sau nuụi cy vi cỏc mu ngi tỡnh nguyn kho mnh - Khụng phỏt hin nhim mycoplasma v vi khun dung dch t bo thu c sau nuụi cy c mu MS04 v MS05 - Nhúm bnh nhõn ung th phi gm bnh nhõn ung th phi khụng phi t bo nh giai on III, s lng t bo bch cu thu c sau nuụi cy t bo tỏch t mỏu tnh mch ngoi vi hai bnh nhõn ung th phi ln lt l 5.796 ì 109 v 1.904 ì 109 5.2 Bc u ỏnh giỏ tớnh ỏp ng dch ca t bo lympho tỏch t bnh nhõn ung th phi sau nuụi cy hot húa - T l t bo lympho T (CD3+) trung bỡnh qun th t bo lympho T, lympho B v NK thu c sau nuụi cy l 95.27%, t l t bo 57 lympho TCD8+ trung bỡnh v lymho TCD4+ trung bỡnhln lt l 52.06 4.497% v 34.83 1.287% - Khụng phỏt hin s tng biu hin mRNA IL-2 v TNF cỏc t bo thu c sau nuụi cy cỏc mu bnh nhõn ung th phi - Khụng phỏt hin nhim mycoplasma v vi khun c dung dch t bo thu c sau nuụi cy c hai mu bnh nhõn ung th phi TI LIấU THAM 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TO B Y T TRNG I HC Y H NI *** O MNH PHNG NuôI cấy hoạt hóa đánh giá tính đáp ứng miễn dịch tế bào lympho tách từ bệnh nhân ung th phổi Chuyờn ngnh : Húa sinh y hc LUN VN BC S NI TR... t bo lympho tỏch t bnh nhõn ung th phi Bc u ỏnh giỏ tớnh ỏp ng dch ca t bo lympho tỏch t bnh nhõn ung th phi ó hot húa 12 CHNG TễNG QUAN TI LIấU Tng quan chung v ung th phi 1.1 Khỏi nim ung th... Tng quan v liu phỏp dch iu tr ung th núi chung v ung th phi 1.2.1 S i ca liu phỏp dch iu tr ung th núi chung v ng dng ung th phi 1.2.1.1 S i ca liu phỏp dch iu tr ung th: Nm 1891, William Coley,