AP alkaline phosphatase ATP adenosine triphosphate BCIP 5-bromo-4-chloro-3-indolyl- phosphate camp cyclic adenosine monophosphate CBAVD congenit

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2 AP alkaline phosphatase ATP adenosine triphosphate BCIP 5-bromo-4-chloro-3-indolyl- phosphate camp cyclic adenosine monophosphate CBAVD congenital bilateral absence of the vas deferens CF cystic fibrosis CFTR cystic fibrosis transmembrane conductance regulator CUAVD congenital unilateral absence of the vas deferens DAB diaminobenzidine DPC Diphenylamine-2-carboxylate E 2 estrogen ENaC epithelial Na conductance FBS fetal bovine serum FSH follicle stimulating hormone FSK forskolin GAPDH glyceraldehydes phosphate dehydrogenase HBSS Hank s blanced salt solution Hank's LH luteinizing hormone IL-1 interleukin-1-1 IOD integrated optical density 1

3 mrna messenger ribonucleic acid MSD membrane-spanning domain NBD nucleotide binding domain NBT nitrobenzene thiocyanate PAF platelet activiting factor PBS phosphate buffer saline PGE 2 prostaglandings E 2 E 2 PGF 2 prostaglandings F 2 F 2 P progesterone PI pancreatic insufficiency PMSF Phenylmethylsulfonyl fluoride RT-PCR reverse transcription-polymerase chain reaction SDS sodium decdecyl sulfate SP streptavidin-peroxidase - SSC sodium chloride/sodium citrate buffer TBS tris-buffered saline Tris TEMED N,N,N,N -tetra methy lethylene diamine N,N,N,N - Tris trishydroxymethylaminomethane 2

4 camp (CFTR) CFTR CFTR western blotting CFTR mrna 1 CFTR mrna, 2 CFTRmRNA 3 CFTR mrna, western blotting 170KDa CFTR RT-PCR CFTR CFTRmRNA CFTR CFTRmRNA CFTRmRNA CFTR mrna CFTRmRNA CFTR CFTR CFTR CFTR forskolin( ) forskolin 3

5 DPC CFTR camp camp CFTR PGE 2 forskolin PGF2 CFTR [ ] camp 4

6 ABSTRACT BACKGROUND: As a camp-regulated Cl - channel, cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in the active secretion of electrolytes and fluid in epithelial cells. Women with CFTR gene mutations are less fertile generally assumed to cervical factors. However, there is little known about CFTR protein expression in human endometrium and its possible roles in reproduction. METHODS AND RESULTS: CFTR protein and mrna levels in human endometrium were analyzed using immunohistochemical and in situ hybridization methods respectively. Significant expression of CFTR protein was only seen in the glandular cells from late-proliferative to all secretory phases, consistent with western blot analysis. While CFTR mrna was presented only around ovulatory period with high levels. In cultured glandular cells, the productions of CFTR protein and mrna were all stimulated by estradiol and inhibited by progesterone. Forskolin-activated Cl - current in endometrial epithelial cells with a linear I/V relationship was detected by whole-cell patch clamp technique. The stimulated channel activities were suppressed by DPC, a CFTR channel blocker. Furthermore, PGE 2 -activated Cl - current also was detected. However the current magnitude induced by PGE 2 was significantly reduced than that stimulated by forskolin. CONCLUSIONS: (1) CFTR mrna and protein localized in human endometrial epithelial cells and the amounts were varied cyclically; (2) CFTR expression in cultured glandular cells was up- and down- regulated by estradiol and progesterone respectively; (3) CFTR in human endometrium functions as a camp-activated Cl - channel. It can be regulated by PGE 2 and play an important role in modulating ion composition of human uterine fluid. 5

7 In summary, the present work was the first to demonstrate the expression of CFTR protein and mrna was cyclic dependent in human endometrium during the menstrual period. The expression of functional CFTR in human endometrial epithelium was thought to be associated with the composition of human uterine fluid. The abundant CFTR mrna and protein around the ovulatory period may drive Na + and fluid from plasma into the uterine lumen to make optimal electrolyte composition and sufficient fluid volume for sperm migration and capacitation. In addition, continuable expression of CFTR proteins has been detected in all secretory phase. Prostaglandin E 2 released by gametes or embryos could activated CFTR channel and thereby inducing chloride ion and water across the endometrial epithelium, it is believed to benefit embryo implantation. Further research of CFTR regulation may improve our knowledge about human reproductive physiology and be helpful to facilitate embryo implantation in treatment of infertile patients by using assisted reproductive technology. [Key words] cystic fibrosis transmembrane conductance regulator; endometrium; camp- activated chloride channel; patch clamp 6

8 cystic fibrosis transmembrane conductance regulator CFTR camp CFTR cystic fibrosis,cf 95 98% [1,2] CF [3] CFTR CFTR CFTR [4] 1989 Riordan CF [5] 7q31 250kb 27 cdna 6129bp kDa ATP (ATP-binding cassette) (motif), membrane-spanning domain MSD nucleotide binding domain, NBD, R- (regulatory domain,rd) R 1991 [6,7,8] CFTR camp 6-10ps, 7

9 - I-V, Br - Cl - >I - >F -, [9] CFTR CFTR CFTR PDZ PDZ [10,11] CFTR [12,13] CFTR 5 [14] CFTR CFTR R ATP NBD Carson 8

10 [15] : camp A(PKA) R ATP NBD C3 NBD1 ATP (O1) NBD2 ATP [16] NBD NBD ATP NBD ADP, NBD ATP 3 CFTR CFTR CFTR camp, camp CFTR, CFTR epithelial Na conductance, ENaC CFTR [17,18] CFTR 9

11 [19] 1 PKA [20] ; 2 CFTR ENaC [21] ; 3 ENaC [22], CFTR CFTR/ENaC Devidas CFTR ATP [23] ATP G CFTR [24] [25,26] [27] GTP- CFTR CFTR [28,29] CFTR CF CFTR camp [30] Ajonuma CFTR [31] CFTR CFTR camp,, \, 10

12 ( ) 30 [32] 2000 CF 1/26 [33] CF 1/3500 [34] 1/15876 [35] 1/9 [36] CFTR CFTR 1000 (The Cystic Fibrosis Genetic Analysis Consortium; F508, (1) F508, 10 3, 508 CF, CF 70 (2) F508,, 40 (3) 1 2 (4) 20 (5) 10 (6), intron8 5T,, [37], CFTR, CFTR NBF 9,10,11,12 19,20,21, 3,4,7 17b, NBF1 11, 5 11

13 ( )CF DeltaF508 (1/n) >0.5%) percent Finland Lucotte et al. [1995]; Kere et al. [1994] Mexico Grebe et al. [1994] Sweden Lucotte et al. [1995]; Schaedel et al. [1999]; Poland Romeo et al. [1989] North Ireland Lucotte et al. [1995] Russia Romeo et al. [1989] Denmark Lucotte et al. [1995] Norway Lucotte et al. [1995] Netherlands Lucotte et al. [1995] Spain Lucotte et al. [1995]; Collee et al. [1998] Greece Lucotte et al. [1995] Germany Lucotte et al. [1995] United States Lucotte et al. [1995]; Romeo et al. [1989] Czech Republic Kosorok et al. [1996]; Romeo et al. [1989] United Kingdom Lucotte et al. [1995]; Romeo et al. [1989] Australia Lucotte et al. [1995]; Romeo et al. [1989] Italy Romeo et al. [1989] France Lucotte et al. [1995]; Gasparini et al. [1999]; Switzerland Romeo et al. [1989] Ireland Lucotte et al. [1995] 12

14 CFTR : CFTR [38], 5 1 CFTR,, 7% CF ; 2 CFTR, CF, F508, CF 85 ; 3 CFTR, camp,, 3% CF ; 4 ; 5, mrna CFTR,, CFTR CF : CFTR, [39], (pancreatic insufficiency, PI) [40], severe mutation mild mutation 13

15 CFTR pancreatic sufficiency, PS CF F508 CFTR >100mmol/L CF CF CF [41] CF CFTR CFTR ( ) CFTR 1993 Trezise CFTR [42] Tizzano CFTRmRNA [43], CFTR CFTR,cAMP Cl - [44],, [45], [46], [47], [48], [49], CFTR CFTR,, CFTR CFTR [50] CFTR 14

16 [43] Sertoli Leydig Sertoli Sertoli CFTR [51] CFTR CFTR Intron8 5T CF Boockfor Sertoli CFTRmRNA [52] camp CFTR ( ) CFTR 1972 Taussig CF [53] CF 1 CFTR (congenital absence of the vas deferens, CAVD) 6% 14% CAVD 18% 50% [54] congenital bilateral absence of the vas deferens, CBAVD 15

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