50% 4 rofecoxib 4 Leydig EDS 2 B COX-2 P < EDS 4 COX-2 mrna EDS 4 COX-2 COX-2 COX-2 COX-2 COX-2 COX-2-2 2

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1 2 2 Cyclooxygenase-2 COX-2 COX-2 COX-2 COX-2 COX-2 COX-2 RT-PCR Western-blot mrna COX-2 A COX-2 Rofecoxib B Leydig EDS 2 4 RT-PCR COX COX-2 COX-2 COX-2 COX-2 mrna COX-2 COX-2 COX-2 Leydig COX-2 rofecoxib 2 A 1

2 50% 4 rofecoxib 4 Leydig EDS 2 B COX-2 P < EDS 4 COX-2 mrna EDS 4 COX-2 COX-2 COX-2 COX-2 COX-2 COX-2-2 2

3 EXPRESSION AND FUNCTION OF CYCLOOXYGENASE-2 IN THE TESTIS MD Candidate: Liu Xiaohui Supervisor: Prof. Xue Zhaoying Co-Supervisor: Prof. Jin Jie The Institute of Urology, Peking University Abstract The cyclooxygenase COX catalyze the conversion of arachidonic acid to PGH 2 which is an obligate precursor of the synthesis of prostaglandins. Recent studies indicate that cyclooxygenase-2 (COX-2) not only plays a central role in inflammation by up-regulating the synthesis of prostaglandins, but also mediates many critical physiological functions by other mechanisms. Despite evidence implicating prostaglandins as critical factor in male reproductive system, little is known about the expression and function of COX-2 in the testis. By using immunohistochemistry, RT-PCR and Western-blot, three sets of experiments were performed. The expression and localization of COX-2 in mature rat and human testis were examined. The changes of spermatogenesis and testosterone concentration in testis were investigated 2, 4 and 8 weeks after administration of the COX-2 specific inhibitor,rofecoxib. To explore the relationship between the intratesticular concentration of testosterone and expression of COX-2 by completely destroyed the Leydig cells of mature male rats with injection of a single I.P. dose of ethane dimethanesulphonate(eds). Four weeks after EDS treatment, the external testosterone was given to the animals. The changes of COX-2 expression and testosterone concentration in testis were detected on the 2nd, 4th, 6th, 8th weeks by methods of immunohistochemistry and RT-PCR. The results showed that COX-2 mrna and protein existed in the mature male rat testis. COX-2 was localized in the seminiferous tubules by immunohistochemistry. COX-2 also existed in adult man testis. COX-2 3

4 protein was positively stained in the seminiferous tubules and the intertubule tissue. Two weeks after administration of rofecoxib, the concentration of testosterone in the whole testis reached its lower values, being only 50% of control values. However, testosterone concentration increased on the 4th week. After such treatment, histologic examination of these testes showed atrophy of the seminiferous tubules and maturation arrest of spermatogenesis. Two weeks afte administration of EDS, the expression of COX-2 decreased significantly (P < 0.005), in comparison with vehicle-treatment controls. Following using external testosterone, though expression of COX-2 mrna and protein is enhanced on 8th week, it wasn t significantly higher than control group. The present study suggests that COX-2 exist in adult mam testis and mature male rat testis. It may be concerned with synthesis of androgen and spermatogenesis in testis. Furthermore, the expression of COX-2 is possibly regulated by androgen, they are positive correlated. Their mechanisms deserve further study. key words Cyclooxygenase-2; Testis; Testosterone; Spermatogenesis; Prostaglandins 4

5 ( ) -BME - Bp Base pair BSA Bovine serum albumin cdna Complementary deoxyribonucleic acid DdH 2 O double distilled water DDT dithiothreitol DEPC diethyl pyrocarbonate DMSO Dimethyl sulfoide EB Ethidium bromide EDS ethane dimethanesulphonate OD Optic density PBS phosphate- buffered saline PCR polymerase chain reaction PI Popidium iodide PKB protein kinase B B rpm rotation per minute RT-PCR reverse transcription polymerase chain reaction Rofecoxib SDS sodium dodecyl sulphate SDS-PAGE SDS-polycrylamide gel electrophoresis SDS- TEMED N,N,N,N,-tetraethyl ethylene diamine N,N,N,N,- Tris tris hydroxy methyl aminomethane 5

6 1 3 5 COX COX COX COX COX COX

8 2 cyclooxygenase,cox prostaglandin H synthase,pghs COX COX-1 COX-2 COX-1 1 (housekeeping functions) COX-2 COX COX-2 COX (Prostaglandins,PGs) 1933 Goldblatt A2/ C 2 COX 2 O 2 a) PGG 2 b) PGG 2 PGH 2 3 PGH 2 PGI 2 PGE 2 PGF 2 A2 TXA 2 ( ) 7,8 8

9 COX G- G E2 EP1-4 PPAR PPAR PPAR PPAR EP2 EP4 camp EP1 EP3 Ca 2+ ( ) NO COX 9

10 COX 3 PG PG 10 COX 2. COX 1971 Vane NSAID COX Miyamoto -1 COX-1 PGHS Herschman Simmons COX COX-1 COX-2 PGHS COX-1 COX COX COX-2 COX COX-1 COX-1 COX-2 mrna COX-2 COX-1 COX-1 PLA 2 (spla 2 ) COX-1 Herschman spla 2 COX-1 18 COX-1 COX COX-2 COX-2 1 1q , 8.3kb 10 9 COX-1 9 9q kb COX-2 COX

11 COX kb 6kb 2.5kb 3 UTR 5 25bp TATA box C/EBP AP-2 SP1 NF-κb CRE Est-1, COX-2 ( ) 19 COX-2 21 COX-2 ORF CTGCGATGC 10 COX-1 COX-2 TIS10 10 COX-1 TIS10 COX-1 COX %-75% 3 38% 19 COX pb 2550bp 3 UTR 3 10 COX-1 COX-2 mrna 3 1.5kb 22 AUUUA mrna COX-2 RNA COX-2 COX-1 COX-2 COX-2 camp 11

12 COX-1 COX-1 14,15 COX-2 COX-2 1kb COX-1 2.4kb C COX-2 63% COX-2 TATAbox COX-1 Sub-baramaiah src ras COX src ras Xie v-src COX-2 CRE Dixon COX-2 mrna COX-2 COX-2 3 AU COX-2 COX-2 mrna 2.4 COX-2 COX-2 PDGF TNF TGF LH-1 TPA PMA v-src ras ET HETES dmpge2 COX-2 COX-2 mrna COX-2 COX NSAID S NS-398 COX-2 P38 MAPK COX 25 G TPA C v-src pp60 v-src COX-2 12

13 2.5 COX-2 COX COX-1 61% 21 COX-2 N COX-1 COX-2 C 18 NASSSRS-GLDDINPTVLLK COX-2 COX-1 18 COX-2 COX-1 COX-2 75% His Try His C Ser COX cdna COX-2 72kD 74kD SDS-PAGE 72kD COX-2 COX kD COX COX 1994 Picot X- COX COX COX 13

14 N C COX NSAIDs 26 COX 2.6 COX 19 COX-1 COX-2 21 COX COX-2 COX RT-PCR COX-2 COX-2 mrna COX-2 mrna 27 COX-2 mrna 2.7 COX COX PGG 2 COX PGG 2 1 C 13 H+ C 11 2 C 11 C 9-11 C 8 3 C 8 C 12 C 15 C 15 C ,11,14 PGG

15 COX-2 1. COX-1 29 COX-1 COX-1 COX-1 30 NSAIDs aspirin PGI 2 COX-1 COX-1 31 NSAIDs 32 NSAIDs COX COX-1 2. COX-2 COX NSAIDs COX-1 COX COX-1 COX-2 COX

16 COX-1 COX-2 COX COX-2 E2 40 Henle COX-1 COX COX-1 COX-2 COX NSAIDs COX COX-2 COX-2 NSAIDs COX-2 LPS Cao COX COX-2 COX-2 45 IL-1 COX COX-2 COX-2 LPS 16

标题

环球中医药 2016 年 5 月第 9 卷第 5 期摇 Global Traditional Chinese Medicine, May 2016,Vol 郾 9, No 郾 5 摇 557 两种电针对脊髓损伤 14 天后大鼠运动功能神经元及 MEK2 p 鄄 ERK1 表达的影响论著宋良玉摇吕威摇景泉凯摇莫雨