本文選題：脂多糖 + 生殖功能��； 參考：《南方醫(yī)科大學》2014年碩士論文

【摘要】：研究背景 近年來,隨著社會的發(fā)展,環(huán)境惡化和人們不良生活習慣影響,男性生育力呈下降趨勢,主要表現在人類的精液質量包括精子濃度和精子活力等指標在逐漸下降。不育癥患病人群不斷增長,也越來越受到人們的關注。不育癥是一種影響人類生存、繁衍的疾病,根據有關部門統(tǒng)計,我國約有10%育齡夫婦存在不育問題,其中一半是由于男方因素引起。男性不育癥成為男科學領域重要的研究課題。 導致男性不育的因素眾多,如先天性生殖器官發(fā)育異常、遺傳疾病、內分泌疾病、生殖系統(tǒng)感染和炎癥以及理化因素等,其中生殖道感染和炎癥與男性不育密切相關,是男性不育的重要致病因素之一,也是當前研究熱點之一。男性生殖道炎癥能夠影響睪丸、附睪等部位,從而在精子生成和發(fā)育階段對精子造成一定的影響。盡管已有大量關于生殖道炎癥與男性不育相關研究,然而炎癥如何發(fā)展并影響男性生育及其可能的機制尚不完全明了。 脂多糖(Lipopolysaccharide,LPS)是革蘭陰性菌細胞壁的主要組成成分,也是革蘭陰性菌的主要致病因子,已被廣泛用于制造動物炎癥模型,研究炎癥對機體的作用。體內注射LPS可產生類似感染的炎癥反應,抑制動物睪丸類固醇激素生成,影響精子發(fā)生,也可導致血-睪屏障損傷,引起抗精子抗體產生,并誘導精子和生精細胞凋亡等,從而損害生育。我們給予雄性大鼠腹膜內注射LPS誘導炎癥反應,觀察了LPS作用后大鼠睪丸組織病理學改變和附睪精子質量變化,并在此基礎上調查了LPS誘導的炎癥對大鼠附睪氧化應激狀態(tài)、睪丸生精細胞凋亡率和血清內分泌激素水平的影響。 有機陽離子/肉堿轉運蛋白2(Organic cation/carnitine transporter2,OCTN2)是左卡尼汀(L-carnitine, LC)的主要轉運載體。附睪上皮細胞基底膜上的OCTN2將血液中的LC主動轉運到附睪腔內,使得LC在附睪液中的濃度比血漿中濃度高出2000倍以上。LC又稱左旋肉堿,是廣泛存在于體內的一種類維生素物質,作為脂肪酸的運輸載體,將長鏈脂肪酸從線粒體膜外轉移到膜內,使其在線粒體基質中進行β-氧化,為人體提供所需要的能量-ATP,促進脂肪酸的代謝。在附睪中,LC運送長鏈脂肪酸進入精子,氧化供能,為精子提供動力,可啟動精子運動、促進精子成熟和提高精子受精能力。精漿LC水平與精子濃度、活動率、正常形態(tài)精子百分率等精液參數之間存在相關性。鑒于LC在男性生殖系統(tǒng)中的重要作用,那么若OCTN2表達改變,將會導致LC轉運障礙,精漿LC水平降低,影響精子成熟。因此,我們調查了LPS作用后大鼠附睪組織OCTN2表達改變。本課題利用LPS誘導雄性大鼠炎癥反應,探索了LPS誘導的炎癥對雄性大鼠生殖功能的影響并初步在分子水平探索其可能的機制。 目的 1.給予雄性SD大鼠腹膜內注射LPS,誘導炎癥反應,觀察大鼠睪丸組織病理學改變和附睪精子質量變化,探討LPS誘導炎癥對雄性大鼠生殖功能的影響； 2.在1的基礎上,調查大鼠附睪氧化應激狀態(tài)、睪丸生精細胞凋亡率和血清T、LH、FSH水平,并進一步研究大鼠附睪組織OCTN2表達改變,探討LPS誘導的炎癥影響雄性大鼠生殖功能的可能機制； 3.通過以上研究,探討臨床生殖道感染和炎癥損害男性生育的相關機制,以期最終實現將研究成果進行轉換,為臨床不育癥診療提供實驗和理論依據。 方法 36只雄性SD大鼠(400～450g)隨機均分為4組：A組(對照組)腹膜內注射無菌生理鹽水；其余3組腹膜內注射LPS (5mg/kg,溶于無菌生理鹽水中),分別于給藥12h(B組)、24h(C組)和72h(D組)后麻醉處死。取左側附睪稱重后,采用WLJY-9000型偉力數碼彩色精子質量檢測系統(tǒng)檢測附睪頭、尾精子活動率,以精子總活動率計算精子活動率；用血細胞計數板按常規(guī)方法計算附睪尾精子數目,并計算精子相對計數值(106個/100mg附睪重)；取大鼠左側睪丸,制備睪丸組織切片,HE染色后,鏡下觀察睪丸組織病理學變化；用硫代巴比妥酸比色法檢測附睪組織勻漿中丙二醛(MDA)含量,黃嘌呤氧化酶比色法測定附睪組織勻漿中超氧化物歧化酶(SOD)活性來反映附睪氧化應激狀態(tài)；流式細胞術檢測睪丸生精細胞凋亡率；酶聯免疫分析法(ELISA)檢測血清睪酮(T)、黃體生成素(LH)和卵泡刺激素(FSH)水平反映生殖內分泌激素改變；提取大鼠附睪組織總RNA, Real-time RT-PCR技術檢測大鼠附睪OCTN2mRNA表達水平變化；提取大鼠附睪組織總蛋白,Western blot技術檢測大鼠附睪OCTN2蛋白表達水平變化。 結果 1.一般情況 給藥前,大鼠飲食正常,狀態(tài)良好。給予LPS作用后約2h開始,大鼠出現寒戰(zhàn)、身體扭曲、波狀緣毛、口鼻分泌物增多和嗜睡等現象,其炎癥反應明顯,4h后狀態(tài)逐漸恢復。 2.大鼠附睪精子計數和精子活動率變化 與A組大鼠附睪頭、尾精子活動率[分別為(62.65±3.61)%和(68.01±1.80)%]相比,B組大鼠附睪頭、尾精子活動率[分別為(56.84±2.65)%和(62.28±4.06)%]明顯降低(P0.05),而C組大鼠附睪頭、尾精子活動率[分別為(37.28±4.97)%和(45.35±3.39)%]以及D組大鼠附睪頭、尾精子活動率[分別為(24.64±4.78)%和(34.85±4.42)%]極顯著降低(P0.01)；與A組大鼠附睪尾精子相對計數[(38.94±4.08)x106個/100mg]相比,B組大鼠附睪尾精子相對計數[(37.15±2.54)×106個/100mg]減少,但無統(tǒng)計學差異(P0.05),而C組[(31.97±2.81)×106個/100mg]和D組[(28.60±4.03)×106個/1OOmg]大鼠附睪尾精子相對計數極顯著減少(P0.01)。 3.大鼠睪丸組織HE染色結果 HE染色鏡下觀察顯示,A組大鼠睪丸生精小管結構清晰,各級生精細胞排列整齊,生精小管管腔內精子數目較多,無脫落的生精細胞；B組大鼠睪丸生精小管結構清晰,各級生精細胞排列基本整齊,部分生精小管管腔內精子數目有所減少,并可見脫落的生精細胞；C組大鼠睪丸生精上皮變薄,生精小管結構較紊亂、不規(guī)則,各級生精細胞減少且排列不整齊,生精小管管腔內精子數目減少,并可見脫落的生精細胞；D組大鼠睪丸生精上皮變薄,生精小管結構較紊亂、不規(guī)則,呈明顯腫脹狀態(tài),各級生精細胞層次紊亂,生精小管管腔內精子數目明顯減少,可見較多脫落生精細胞阻塞管腔。 4.大鼠附睪組織氧化應激狀態(tài)改變 硫代巴比妥酸比色法檢測結果顯示,與A組大鼠附睪組織勻漿中MDA含量[(4.66±1.49)nmol/mgprot]相比,B組[(15.95±3.26)nmol/mgprot]和C組[(12.93±2.54)nmol/mgprot]大鼠附睪MDA含量極顯著升高(P0.01),而D組大鼠附睪MDA含量[(9.67±1.68)nmol/mgprot]明顯升高(P0.05)；黃嘌呤氧化酶比色法測定結果顯示,與A組大鼠附睪組織勻漿中SOD活性[(879.335±105.089)U/mgprot]相比,B組大鼠附睪組織勻漿中SOD活性[(729.265±93.783)U/mgprot]降低,但無統(tǒng)計學差異(P0.05),C組[(694.126±58.530)U/mgprot]和D組[(655.352±115.215)U/mgprot]大鼠附睪組織勻漿中SOD活性顯著降低(P0.05)。 5.大鼠睪丸生精細胞凋亡率變化 流式細胞術結果顯示,與A組大鼠睪丸生精細胞凋亡率(4.21±1.67)%相比,B組大鼠睪丸生精細胞凋亡率(11.01±3.30)%明顯增加(P0.05),而C組(23.88±4.58)%和D組(41.28+2.28)%大鼠生精細胞凋亡率極顯著增加(P0.01)。 6.大鼠血清T、LH和FSH水平改變 ELISA檢測結果顯示,與A組大鼠血清T水平[(0.490±0.028)ng/ml]相比,B組大鼠血清T水平[(0.460±0.024)ng/ml]降低,但無統(tǒng)計學差異(P0.05),而C組[(0.417±0.021)ng/ml]和D組[(0.378±0.021)ng/ml]大鼠血清T水平極顯著降低(P0.01)；與A組大鼠血清LH水平[(6.290±0.515)ng/L]相比,B組大鼠血清LH水平[(5.881±0.124)ng/L]降低,但無統(tǒng)計學差異(P-0.05),而C組[(5.123±0.271)ng/L]和D組[(4.504±0.279)ng/L]大鼠血清LH水平極顯著降低(P0.01);與A組大鼠血清FSH水平[(1.837±0.127)IU/L]相比,B組大鼠血清FSH水平[(1.707±0.098)IU/L]降低,但無統(tǒng)計學差異(P0.05),而C組[(1.620±0.115)IU/L]和D組[(1.562±0.216)IU/L]大鼠血清FSH水平顯著降低(P0.05)。 7.大鼠附睪組織OCTN2表達改變 Real-time RT-PCR檢測結果和Western blot檢測結果相符：與A組大鼠附睪組織OCTN2mlRNA表達量(1.00±0.00)相比,B組大鼠附睪OCTN2mRNA表達量(0.962±0.016)降低,但無統(tǒng)計學差異(P0.05),而C組(0.706±0.026)和D組(0.474±0.037)大鼠附睪OCTN2mRNA表達量極顯著降低(P0.01)；與A組大鼠附睪組織OCTN2蛋白表達量(1.707±0.092)相比,B組大鼠附睪OCTN2蛋白表達量(1.458±0.165)降低,但無統(tǒng)計學差異(P0.05),而C組(1.197±0.118)和D組(1.023±0.136)大鼠附睪OCTN2蛋白表達量極顯著降低(P0.01)。 結論 1. LPS (5mg/kg)腹膜內注射誘導大鼠炎癥反應,導致大鼠附睪精子計數和精子活動率降低以及睪丸組織病理學改變,影響大鼠生殖功能； 2.LPS誘導的炎癥反應破壞大鼠附睪氧化應激狀態(tài)、增加大鼠睪丸生精細胞凋亡率并降低血清T、LH、FSH水平,這些指標改變可能是LPS誘導炎癥損害生殖功能的相關因素； 3.LPS誘導的炎癥反應抑制大鼠附睪組織OCTN2表達,可能是LPS誘導炎癥損害雄性大鼠生殖功能的機制之一。
[Abstract]:Background of the study

In recent years , with the development of society , the deterioration of environment and people ' s bad habits , the fertility of male is decreasing . The quality of human semen includes such indexes as sperm concentration and sperm motility .

Male infertility has many factors , such as abnormal reproductive organs development , genetic diseases , endocrine diseases , reproductive system infection , inflammation and physical and chemical factors , among which reproductive tract infection and inflammation are closely related to male infertility , which is one of the important pathogenic factors of male infertility .

lipopolysaccharides ( LPS ) is the main component of the cell wall of Gram - negative bacteria , and is the main pathogenic factor of Gram - negative bacteria , and has been widely used in the manufacture of animal inflammation models .

This study investigated the effects of LPS on the reproductive function of male rats and explored the possible mechanism of LPS - induced inflammation in male rats .

Purpose

1 . LPS was injected intraperitoneally to male SD rats , inflammatory reaction was induced , pathological changes of rat testis were observed , and the changes of sperm quality were observed , and the effects of LPS - induced inflammation on the reproductive function of male rats were investigated .


2 . On the basis of 1 , the oxidative stress state , apoptosis rate and serum T , LH and FSH levels were investigated in rats , and the expression of OCTN2 in the testis of rats was further studied , and the possible mechanism of LPS - induced inflammation on the reproductive function of male rats was discussed .


3 . Through the above research , the relative mechanism of reproductive tract infection and inflammation is discussed in order to realize the transformation of the research results and provide the experimental and theoretical basis for the diagnosis and treatment of clinical infertility .

method

36 male SD rats ( 400 - 450 g ) were randomly divided into 4 groups : group A ( control group ) intraperitoneal injection of sterile physiological saline ;
In the remaining three groups , LPS ( 5 mg / kg , dissolved in sterile physiological saline ) was injected intravenously for 12 h ( group B ) , 24 h ( group C ) and 72 h ( group D ) .
The number of sperm count was calculated by routine method by blood cell counting plate , and the relative count value of sperm was calculated ( 106 / 100 mg / 100 mg ) ;
The testis of the left side of the rat was obtained , the testis tissue sections were prepared , and the pathological changes of the testis were observed under the microscope after HE staining ;
The content of malondialdehyde ( MDA ) in the homogenate of the tissue homogenate was determined by thiobarbituric acid colorimetry , and the activity of superoxide dismutase ( SOD ) in the homogenate of the tissue homogenate was determined by xanthine oxidase colorimetric method .
The apoptosis rate of spermatogenic cells was detected by flow cytometry .
Serum testosterone ( T ) , LH and FSH levels were measured by enzyme linked immunosorbent assay ( ELISA ) .
The expression level of OCTN mRNA was detected by real - time RT - PCR in rats with total RNA and Real - time RT - PCR .
The expression level of OCTN2 protein was detected by Western blot .

Results

1 . General situation

Before administration , the rats had normal diet and good condition . After administration of LPS for about 2 hours , the rats appeared cold war , body torsion , undulate margin , mouth - nasal discharge increased and drowsiness , etc . , their inflammatory responses were obvious , and the state gradually recovered after 4h .

2 . Changes of sperm count and sperm motility in rats

Compared with group A , the activity of sperm motility was ( 56.84 鹵 2.65 ) % and ( 62.28 鹵 4.06 ) % , respectively ( 56.84 鹵 4.97 ) % and ( 45.35 鹵 3.39 ) % and ( 34.85 鹵 4.42 ) % , respectively ( P < 0.01 ) .
Compared with group A , the relative counts of sperm in tail sperm of rats ( 37.15 鹵 2.54 ) 脳 106 / 100 mg / kg were significantly decreased ( P < 0.01 ) , but there was no statistical difference ( P < 0.01 ) .

3 . HE staining results of rat testis

Under HE staining , the structure of spermatogenic tubules was clear in group A , and the spermatogenic cells at all levels were arranged in order , and the number of spermatogenic cells in the tubules of spermatogenic tubules was more than that of spermatogenic cells .
In group B , the structure of spermatogenic tubules was clear , the arrangement of spermatogenic cells at all levels was basically regular , the number of sperm in the tubules of partially spermatogenic tubules decreased , and the spermatogenic cells shed were observed .
In group C , the spermatogenic epithelium of the testis was thinner , the structure of spermatogenic tubules was disordered , irregular , the spermatogenic cells at all levels decreased and the arrangement was irregular , the number of spermatogenic cells in the tubules of spermatogenic tubules decreased , and the detached spermatogenic cells were observed ;
In group D , the spermatogenic epithelium of the testis was thinned , the structure of spermatogenic tubules was disordered , irregular , and the level of spermatogenic cells at all levels was disordered , and the number of sperm cells in the tubules of spermatogenic tubules was significantly decreased .

4 . Changes of oxidative stress state in the rat testis

Compared with group A , the content of MDA was significantly higher in group B than that in group A ( 15.95 鹵 3.26 ) nmol / mg in group B ( 12.93 鹵 2.54 ) nmol / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg , respectively , compared with that in group A ( P < 0.01 ) .
Compared with group A , the activity of SOD decreased ( 729,265 鹵 93.783 ) U / mgT lymphocyte decreased , but there was no statistical difference ( P0.05 ) .

5 . Apoptosis rate of spermatogenic cells in rat testis

Compared with group A , the apoptosis rate of spermatogenic cells ( 11.01 鹵 3.30 ) % increased significantly ( P < 0.01 ) , while the apoptosis rate of spermatogenic cells in group C ( 23.88 鹵 4.58 ) % and group D ( 41.28 + 2.28 ) % increased significantly ( P0.01 ) .

6 . Changes of serum T , LH and FSH levels in rats

The results of ELISA showed that the serum T level in group B was significantly lower than that in group A ( 0.490 鹵 0.028 ) ng / ml , but there was no statistical difference ( P < 0.05 ) , but the serum T level was significantly lower in group C ( 0.417 鹵 0.021 ) ng / ml and D group ( 0.378 鹵 0.021 ) ng / ml ( P0.01 ) .
Compared with group A , the serum LH levels were significantly lower in group B ( 5.881 鹵 0.124 ) ng / L , but there was no statistical difference ( P - 0.05 ) , but there was no statistical difference ( P - 0.05 ) . The serum FSH levels in group B were significantly lower than those in group A ( 1.620 鹵 0.115 ) IU / L and D group ( 1.562 鹵 0.216 ) IU / L , respectively ( P0.05 ) .

7 . Changes of OCTN2 Expression in the Testis of Rats

The results of real - time RT - PCR and Western blot analysis showed that the expression of OCTN mRNA was decreased in group B rats ( 0 . 962 鹵 0 . 016 ) , but there was no statistical difference ( P 0 . 05 ) , while that of C group ( 0 . 706 鹵 0.026 ) and D group ( 0 . 474 鹵 0 . 37 ) decreased significantly ( P0.01 ) .
The OCTN2 protein expression ( 1.458 鹵 0.165 ) was lower in group B than in group A ( 1.707 鹵 0.092 ) , but there was no statistical difference ( P < 0.05 ) , while the expression of OCTN2 protein in group C ( 1.197 鹵 0.118 ) and group D ( 1.023 鹵 0.136 ) decreased significantly ( P0.01 ) .

Conclusion

1 . Intraperitoneal injection of LPS ( 5mg / kg ) induced inflammatory reaction in rats , which resulted in the decrease of sperm count and sperm motility and pathological changes of testis in rats , which could affect the reproductive function of rats .


2 . LPS - induced inflammatory reaction destroys the oxidative stress state of rat testis , increases the apoptosis rate of rat testis spermatogenic cells and decreases serum T , LH and FSH levels .


3 . LPS - induced inflammatory response can inhibit the expression of OCTN2 in rats , which may be one of the mechanisms of LPS - induced inflammation to damage the reproductive function of male rats .
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R698.2

【參考文獻】

相關期刊論文 前9條

1 董強;精漿抗氧化酶與男性不育[J];國外醫(yī)學.泌尿系統(tǒng)分冊;2001年06期

2 李積勝,閆蓓,劉亞華;一氧化氮合酶抑制劑對嗎啡依賴大鼠睪丸細胞凋亡的影響[J];中華男科學雜志;2004年11期

3 李錚,陳國武,商學軍,白文俊,韓銀發(fā),陳斌,滕曉明,孟凡會,張濱,陳德寧,劉繼紅,鄭新民,曹小蓉,劉勇,朱曉斌,王益鑫;左旋肉堿和乙酰左旋肉堿合用治療少弱精子癥有效性與安全性的多中心隨機對照臨床研究[J];中華男科學雜志;2005年10期

4 李克;李偉;黃宇烽;商學軍;;精漿游離L-肉毒堿水平及其與精子密度、活動率及活力的相關性研究[J];中華男科學雜志;2007年02期

5 龔東明;李錚;朱曉斌;劉玉林;曹小蓉;劉勇;王益鑫;;有機陽離子轉運子2在人類附睪中的表達及其意義[J];中華男科學雜志;2008年03期

6 段東升;;氧自由基與精子功能[J];男性學雜志;1990年02期

7 薛麗英;李杰;王更新;修賀明;孫輝臣;;脂多糖誘導的非特異性炎癥對大鼠睪丸功能的影響[J];生殖與避孕;2006年02期

8 范莉英，，冷雙英，趙春芳，趙巖;切除頜下腺對小鼠睪丸、精子計數和血清睪丸酮的影響[J];生殖與避孕;1996年06期

9 張雪梅;熊煥章;;LPS誘導的炎癥反應信號傳導通路研究進展[J];中國獸醫(yī)雜志;2010年07期

本文編號：1913374