基因重排检测技术的建立及其在淋巴瘤诊断中的应用.pdf

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4、 r r a n g e m e n t s o f Ig H a n d TCR a n d t o a s s e s s t h e a p p Ii c a t i o n o f t h e d e t e c t i o n t e c h n i g u e i n t h e d i a g n o s i s o f m a Ii g n a n t Iy m p h o m a . M ET H O D S F i f t y -t h r e e c a s e s o f m a Ii g n a n t Iy m p h o m a a n d 20 c a s e

5、s o f r e a c t i v e Iy m p h o i d h y p e r p Ia s i a w e r e c h o s e n f o r m a k i n g t i s s u e s e c t i o n s . Ge n o m i c D NA w a s e x t r a c t e d a n d a m p Ii f i e d v i a PCR . Th e p r o d u c t s w e r e r e s o Iv e d o n d e n a t u r i n g p o Iy a c r y Ia m i d e g e

6、 Is a n d v i s u a Ii z e d t h r o u g h s i Iv e r s t a i n i n g . R ES U L T S Ge n e r e a r r a n g e m e n t s o f Ig H a n d TCR w e r e n e g a t i v e i n 20 c a s e s o f r e a c - t i v e Iy m p h o i d h y p e r p Ia s i a . In a II o f 18 c a s e s o f T c e II-n o n - H o d g k i n

7、s Iy m p h o m a T-NH L g e n e r e a r r a n g e m e n t o f TCR ! w a s f o u n d i n 12 c a s e s a n d t h a t o f TCR “ w a s f o u n d i n 3 c a s e s a n d Ig H g e n e r e a r r a n g e m e n t w a s n o t f o u n d . Ig H g e n e r e a r r a n g e - m e n t w a s f o u n d i n 28 c a s e s

8、o u t o f 32 c a s e s o f B c e II-NH L B- NH L i n 2 c a s e s o f w h i c h g e n e r e a r r a n g e m e n t s o f TCR ! a n d Ig H w e r e b o t h f o u n d . Th e r e w a s Ig H g e n e r e a r r a n g e m e n t i n 3 c a s e s o f s u s p e c t e d Iy m p h o m a s . Th e r e w a s a r e m a

9、r k a b Ie d i f f e r e n c e b e t w e e n NH L g r o u p a n d b e n i g n g r o u p i n g e n e r e a r r a n g m e n t P 0 .05 . CO N CL U S I O N Th e d e t e c t i o n o f g e n e r e a r r a n g e m e n t s o f Ig H a n d TCR b y PCR c a n b e a k i n d o f s u b s i d i a r y m e a n s t o

10、d i a g n o s e t h e Iy m p h o p r o Ii f e r a t i v e d i s e a s e . Pa r t i c u Ia r Iy t h e c Io n a Ii t y s h o w e d i n t h e f o r m a Ii n -f i x e d a n d p a r a f f i n -e m b e d d e d s a m p Ie s m a y b e u s e f u I i n t h e r e t r o s p e c t i v e r e s e a r c h e s . K e

11、 y w o r d s Iy m p h o m a g e n e r e a r r a n g e m e n t p o Iy m e r a s e c h a i n r e a c t i o n n e o p Ia s m B T B T . 5 10 D NA PCR . 20 Ig H TCR 18 T TCR ! 12 TCR “ 3 Ig H 32 B 28 Ig H 2 TCR ! Ig H 3 Ig H Ig H TCR P 0 .05 . PCR B Ig H T TCR . R 733 .4 A 0 m a Ii g n a n t Iy m p h o m

12、 a . H o d g k i n s d i s e a s e H D n o n -H o d g k i n s m a Ii g n a n t Iy m p h o m a NH L H E NH L . Ig TCR . T B . 1 . . . 1 1.1 53 1995 20 0 5 . 29 6 7 7 JF o u r t h Mi I Me d Un i v 2006 27 9 h t t p / j o u r n a I.f m m u .e d u .c n 55% 24 45% 12 76 44.2 . . B 32 T 18 3 . 20 . P CR H

13、 E B CD20 CD79a T CD3 CD45R O . 3 B-NH L CD79a . !.“# 1.2.1 DNA 4卜 m 6 . 1.5 m L 2-3 DNA. 1.2.2 . p - P C03 ACACAACTGTGTTCACTAGC P C04 CAACT- TCATCCACGTTCACC Ig H FR 3A ACACGGCTGTGTATTACTGT LJH TGAG- GAGACGGTGACC VLJH GTGACCAGGGTACCTTG- GCCCCAG T TCR p v Db 1 CAAAGCTGTAACATTGTGGGGAC Jb 2 AG- CACTGTG

14、AGCCTGGTGCC T VG AGGGTTGTGTTG- GAATCAGG T J CGTCGACAACAAGTGTTGTTC- CAC V v 11 TCTGGAGTCTATTACTGTGC V v 101 CT- CACACTCTCACTTC J v 12 CAAGTGTTGTTCCACT- GCC Jp 12 GTTACTATGAGCTAGTCC. 1.2.3 P CR P CR P T-200 M J R e Se a r c h . 25 卜 L 10 m m o I/L d NTP Gi b c o BR L 2卜 L 10X 2.5 卜 L 50 m m o I/L M g

15、CI 2 0.75 卜 L !“# DNA Gi b c o BR L 1U 20 p m o I/L 1卜 L DNA 1 5 卜 L. Ig H P CR FR 3A LJH 95C 3 m i n 94C 40 S 62C 50 S 72C 40 S 30 72C 10m i n . 11 100 1卜 L FR 3A VLJH 95C 3 m i n 94C 40 S 60C 50 S 72C 40 S 30 72C 10 m i n . FR 3A VLJH 100 120 b p . TCR p TCR v 40 TCR v V v 11 V v 101 J v 12 V v 11

16、 V v 101 Jp 12 . 95C 3 m i n 94C 40 S 56C 50 S 72C 40 S 72C 10 m i n . TCR p 65 85 b p . TCR v 70 200 b p . p - 40 .95C 3m i n 94C 40S 60C 50 S 72C 40 S 72C 10 m i n . 110 b p . 1.2.4 20 g /L 3卜 L 4 990 m L/L 1 g /L 1 g /L 0.8 m m 100g /L 8m o I/L m i n i VE Am e r - Sh a m -P h a r m a c i a Bi o t

17、 e c h 8 h 80 V . 2 UVP UVP Ca m b r i d g e UK Ig H TCR . 1.2.5 P CR 1 m m 5 b p . SP SS 10.0 ! 2 $ 0.05 . “# “.!# # 32 B CD20 CD79a CD3 CD45R O 1 18 T CD3 CD45R O CD20 CD79a 2 A CD79a B CD20. 1 B-NH L CD79a CD20 7 7 7 J Fo u r t h M i IM e d Un i v 2006 27 9 h t t p /j o u r n a I.f m m u .e d u .

18、c n .3 CD79! CD3 CD45RO B- NH L .20 CD20 CD79! CD3 CD45RO . A CD45RO B CD3 . 2 T- NH L CD45RO CD3 !. !“ 2. 2. l DNA PC03 PC04 DNA l l 0 b p “- 3 . l 00 b p DNA .l T- NH L . l l 0 b p 2 B- NH L 3 T- NH L TCR# 4 B- NH L Ig H 5 . 3 PC 03 /PC 04 PCR 2. 2.2 20 B T 4 . 2. 2. 3 T B B B Ig 5 .T T TCR#l 6 .

19、l 00 b p DNA .l Ig H FR3 A/L JH FR3 A/ VL JH 2 TCR“Db l /Jb 2 3 TCR#l T VG / T JX 4 TCR#2 V #l l /V #l 0l /J #l 2 5 TCR#3 V #l l /V #l 0l /Jp l 2 . l l 0 b p . 4 PCR l 00 b p DNA .l PC 03 /PC 04 2 Ig H FR3 A/L JH FR3 A/VL JH 3 TCR“Db l / Jb 2 4 TCR#l T VG /T JX 5 TCR#2 V #l l / V #l 0l /J #l 2 6 TCR

20、#3 V #l l /V #l 0l /Jp l 2 7 FR3 A/L JH FR3 A/VL JH . l l 0 b p . 5 B- NH L PCR 2. 2. 4 l 8 T TCR# l 2 TCR“ 3 TCR 83 .3 % TCR ! 2 =l 5.l 0 ! 0. 05 32 B 28 Ig H 2 TCR# 87.5% Ig H ! 2 =25.30 ! 0. 05 . 8 7 7 JFo u r t h M i I M e d U n i v 2006 27 9 h t t p /j o u r n a I.f m m u .e d u .c n 100b p D N

21、A . 1 TC R!D b 1/Jb 2 2 TC R“3 V “11 /V “101 /Jp 12 3 TC R“2 V “11 /V “101 / J “12 4 TC R“1 T VG /T JX PC 03/PC 04 5 TC R“1 T VG /T JX PC 03/PC 04 6 TC R“2 V “11 /V “101 /J “12 7 TC R“3V “11 /V “101 /Jp 12 8 Ig H F R3A/LJH F R3A/VLJH . 110b p 190b p 80b p . 6 T-NHL PC R 2.2.5 3 Ig H F R3A/LJH F R3A/VLJH PC R 95 b p Ig H . !“ m a Ii g n a n t Iy m p h o m a . HE