本文關(guān)鍵詞： 桔梗皂苷D 精子 殺精劑 凋亡 避孕 陰道刺激性　出處：《第三軍醫(yī)大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的： 人口爆炸已成為全球重大問題。在全球范圍內(nèi)，每一年有超過2億個妊娠發(fā)生，而其中50%是意外妊娠。我國是人口大國，計劃生育是我們的基本國策，避孕藥的研發(fā)一直備受關(guān)注。殺精劑是用于避孕的藥物，在性交前用于陰道以避免妊娠，殺精劑配合其他屏障性避孕措施使用是一種非常有效的避孕措施。目前廣泛使用的殺精劑壬苯醇醚-9（nonoxynol-9，N-9）可能增加艾滋病感染可能性，因而亟需研發(fā)新的殺精劑。中藥是我們的傳統(tǒng)藥物，是藥物研發(fā)的寶貴資源庫，山東省計劃生育科學(xué)技術(shù)研究所的前期研究從數(shù)百種單味中藥中已發(fā)現(xiàn)桔梗、遠(yuǎn)志、公丁香具有快速體外殺精子活性，其中桔梗皂苷類提取物的殺精活性最強(qiáng)。有研究顯示來自植物的天然皂苷類是一類很有發(fā)展前景的體外殺精劑。本研究通過預(yù)實(shí)驗(yàn)證實(shí)桔梗中含量最豐富的一種皂苷-桔梗皂苷D（Platycodin D，PD）具有體外快速殺精子活性，進(jìn)一步通過與其他數(shù)種皂苷單體比較，證實(shí)PD的體外殺精活性，并通過精子凋亡檢測、低滲腫脹實(shí)驗(yàn)、透射及掃描電鏡等探索其殺精機(jī)制，同時通過DNA損傷實(shí)驗(yàn)、體內(nèi)避孕實(shí)驗(yàn)、陰道刺激實(shí)驗(yàn)等初步研究其成藥可能性，以期望獲得具有殺精作用的臨床前候選藥物。 方法和結(jié)果： 1.伊紅染色檢測精子活性 （遠(yuǎn)志酸或桔梗皂苷D）1μl+伊紅49μl，混勻，取混合液5μl+5μl精液，載玻上混勻，觀察精子著色情況。0.25mmol/L桔梗皂苷D（PD）在瞬間殺滅全部精子（20s），同樣條件下，遠(yuǎn)志酸（Polygalacic acid，PA）殺滅精子所需濃度為7.5mmol/L。PA有效殺精濃度太高被淘汰，0.2mmol/LPD可有效殺滅精子，作為進(jìn)一步實(shí)驗(yàn)的作用濃度。 2.計算機(jī)輔助精子分析系統(tǒng)（CASA）篩選備選藥物并分析目標(biāo)藥物殺精活性 將同一健康男性的精液均分為6份，分別用0.9%NaCl對照，1%DMSO，0.2mmol/l遠(yuǎn)志皂苷B（OnjisaponinB，OB），0.2mmol/L常春藤皂苷B（Hederasaponin B，HB），0.2mmol/L去芹菜糖桔梗皂苷D（Deapio platycodin D，DPD）處理精子2min。用計算機(jī)輔助的精液分析系統(tǒng)（CASA）檢測備選藥物對人精子的殺傷效果。篩選出PD為活性最強(qiáng)的單體，將同一健康男性的精液均分為4份，分別用0.9%NaCl對照，0.15mmol/LPD，0.20mmol/LPD，0.20mg/ml N-9(=0.308mmol/L)陽性對照處理2min。CASA檢測0.15mmol/LPD精子活率（a+b+c%）為47.15±12.44，0.20mmol/LPD精子活率21.38±10.69，0.25mmol/LPD精子瞬間完全失活。得出PD最佳殺精濃度是0.20mmol/LPD，用于后繼實(shí)驗(yàn)。 3.殺精機(jī)制研究 健康男性的精液上游后，用凋亡試劑盒檢測PD對人精子的凋亡誘導(dǎo)作用；用低滲腫脹檢測試劑盒，LIVE/DEAD Sperm Viability Kit檢測PD對人精子細(xì)胞膜的完整性及存活狀態(tài)的影響。結(jié)果發(fā)現(xiàn)0.20mmol/LPD引起大量精子發(fā)生晚期凋亡，精子腫脹率下降，LIVE/DEAD檢測發(fā)現(xiàn)大量死亡精子；掃描和透射電鏡結(jié)果顯示，精子頭頸處斷裂，精子膜破裂，線粒體腫脹及膜丟失； CometAssay結(jié)果顯示0.20mmol/L PD不會對精子DNA造成損傷。 3.1精子凋亡檢測：0.10mmol/L,0.15mmol/L,0.20mmol/L,0.25mmol/L PD使大量精子發(fā)生凋亡，以晚期凋亡和壞死（即FITC+PI+）為主；0.25mmol/L PD晚期凋亡及壞死精子比例占精子總數(shù)的75.84±11.12%；各劑量組PD對精子的早期凋亡（FITC+PI-）均沒有影響。表明PD破壞了精子膜。 3.2低滲腫脹實(shí)驗(yàn)：0.9%NaCl對照組多數(shù)精子發(fā)生了膨脹（尾部卷曲），而PD組精子膨脹不明顯，低滲腫脹反應(yīng)消失，膨脹率從76.5±6.12%下降28.0±6.13%，兩組間膨脹率有顯著差異。結(jié)果顯示精子膜受到PD損傷。 3.3LIVE/DEAD檢測：對照組精子以綠色熒光（活精子）為主，PD處理后橙色熒光（頻死精子）、紅色熒光（死亡精子）明顯增加，陽性對照組（0.2mmol/L N-9）與0.1mmol/L PD組殺精效果相近，0.2mmol/L PD組精子幾乎都是紅色熒光，殺精率95%。 3.4掃描電鏡及透射電鏡：在掃描電鏡下，對照組精子形態(tài)正常，頂體清楚，用藥后，可見大量精子頭頸處斷裂。在透射電鏡下，對照組精子細(xì)胞膜完整，線粒體結(jié)構(gòu)完整。用藥后，，精子膜破裂，線粒體腫脹，甚至膜脫落。 3.5Comet Assay實(shí)驗(yàn)：陰性對照組，PD0.2mmol/L組的所有實(shí)驗(yàn)用精子，均沒有發(fā)現(xiàn)DNA移動尾巴，陽性對照組有明顯的DNA移動尾巴。 4.動物實(shí)驗(yàn) 4.1避孕實(shí)驗(yàn)：雌性SD大鼠右側(cè)子宮注入0.9%NaCl100μl（對照組），在左側(cè)子宮給予3mg/ml PD100μl(劑量為0.3mg)，術(shù)后與雄鼠2:1合籠；第二天發(fā)現(xiàn)陰栓后分籠，10天后處死老鼠，發(fā)現(xiàn)注射PD側(cè)（左側(cè)）子宮無孕囊，而注射生理鹽水側(cè)（右側(cè)）子宮正常妊娠。 4.2陰道刺激實(shí)驗(yàn)：雌性大鼠隨機(jī)分為5組，每天定時給予0.2ml（約0.5g)PD凝膠陰道局部給藥，連續(xù)給藥14天，末次給藥后處死動物，取出陰道組織，用甲醛固定，進(jìn)行組織病理學(xué)檢查及陰道刺激評分[40]。自然對照組，空白凝膠組，3mg/g PD組，8mg/gPD組，8mg/g N-9組的陰道刺激評分分別為2.11±2.01，3.22±1.07，4.89±0.69，7.44±1.58，3.67±1.53。陰道刺激試驗(yàn)評分低于8分，刺激性在可以接受范圍內(nèi)。結(jié)論： PD對人精子有顯著的瞬間殺滅效應(yīng)，能引起精子晚期凋亡，殺精機(jī)制研究顯示PD直接損傷精子膜。殺精濃度的PD對DNA無損傷，大鼠避孕實(shí)驗(yàn)證實(shí)PD在體內(nèi)可有效避孕，對陰道刺激性在可接受范圍內(nèi)。PD將來可能作為一種有潛力的臨床殺精劑用于避孕。
[Abstract]:Background and purpose:
The population explosion has become a major problem in the world. Globally, every year there are more than 200 million pregnancy, of which 50% is pregnant. China is a populous country, family planning is our basic national policy, developing contraceptives has attracted much attention. The spermicidal agent is used for contraceptive drugs for vaginal intercourse in to avoid pregnancy, spermicide with other barrier contraceptive use is a very effective contraceptive measures. The widely used spermicide nonoxynol-9 -9 (nonoxynol-9, N-9) may increase the possibility of HIV infection, and therefore need to develop new spermicide. Traditional Chinese medicine is our drug is. The precious resources for drug development, preliminary study of family planning in Shandong province science and Technology Research Institute from hundreds of Chinese herbs have been found in Platycodon grandiflorum, Polygala tenuifolia, clove has rapid spermicidal activity in vitro, the saponins of Platycodon grandiflorum Extract spermicidal activity was the strongest. Studies have shown that natural saponins from plants is a kind of promising spermicide. This study through the preliminary study confirmed a Platycodon saponins - the most abundant content of platycodin D (Platycodin D PD) has in vitro rapid spermicidal activity, further through compared with several other kinds of saponin monomer, confirmed the spermicidal activity of PD in vitro, and the sperm apoptosis detection, hypoosmotic swelling test, transmission and scanning electron microscopy to explore the spermicidal mechanism, at the same time through the DNA damage test in vivo contraceptive experiment, preliminary study on vaginal irritation experiment to obtain the possibility of medicine, has killed the role of the fine preclinical drug candidates.
Methods and results:
Detection of sperm activity by 1. eosin staining
(Polygalic acid or platycodin D) 1 l+ eosin 49 L, mixing, mixing liquid 5 l+5 l glass loading on semen, mixing, observe the sperm coloring of.0.25mmol/L platycodin D (PD) in an instant kill all sperm (20s), under the same conditions, Polygalic acid (Polygalacic acid, PA) required to kill sperm concentration is 7.5mmol/L.PA effective spermicidal concentration is too high to be eliminated, 0.2mmol/LPD can effectively kill sperm, as the concentration of further experiments.
2. computer assisted sperm analysis system (CASA) screening alternative drugs and analysis of target drug spermatozoon activity
Sharing the same semen in healthy men was 6, respectively, with 0.9%NaCl control, 1%DMSO, 0.2mmol/l Tenuigenin B (OnjisaponinB, OB), 0.2mmol/L B (Hederasaponin B hederin, HB, 0.2mmol/L) to apiose platycodin D (Deapio platycodin D, DPD) system of sperm 2min. using computer assisted semen analysis (CASA) the killing effect of detection of candidate drug on human sperm. Selected from the PD monomer is the most active, sharing the same semen in healthy men for 4 copies, respectively with 0.9%NaCl control, 0.15mmol/LPD, 0.20mmol/LPD, 0.20mg/ml N-9 (=0.308mmol/L) positive control 2min.CASA detection of 0.15mmol/LPD sperm motility (a+b+c%) was 47.15. 12.44,0.20mmol/LPD 21.38 + 10.69,0.25mmol/LPD sperm motility instantly completely inactivated. The PD optimal spermicidal concentration is 0.20mmol/LPD, used for experiments.
3. study on the mechanism of sperm killing
Semen upstream of healthy men, with apoptosis kit for detection of PD induced apoptosis on human sperm; swelling test kit with low permeability, impact on the integrity of human sperm membrane and LIVE/DEAD Sperm Viability Kit to detect the survival status of PD. The results showed that 0.20mmol/LPD induced apoptosis caused by a large number of sperm, sperm swelling rate decreased. LIVE/DEAD detected that the death of a large number of sperm; scanning and transmission electron microscopy results show that the fracture of head and neck of sperm, sperm membrane rupture, mitochondrial swelling and loss of membrane; CometAssay results showed that 0.20mmol/L PD will not cause damage to sperm DNA.
Detection of 3.1 sperm apoptosis: 0.10mmol/L, 0.15mmol/L, 0.20mmol/L, 0.25mmol/L and PD so that a large number of sperm apoptosis in late apoptosis and necrosis (FITC+PI+); 0.25mmol/L PD late apoptosis and necrosis accounted for 75.84 of the total number of sperm sperm + 11.12%; early apoptosis in each dose group PD on sperm (FITC+PI-) showed that PD had no effect. Destroy the sperm membrane.
3.2 hypotonic swelling test: most of the 0.9%NaCl control group had swelling (tail curl), while the PD group had no obvious expansion of sperm and low permeability and swelling reaction disappeared. The expansion rate decreased from 28 to 6.13% of 76.5 + 6.12%, and the expansion rate between the two groups was significantly different. The results showed that the sperm membrane was damaged by PD.
3.3LIVE/DEAD detection: the control group with green fluorescence (sperm live sperm), PD treated orange fluorescence (frequency of dead sperm), red fluorescence (dead sperm) increased significantly, the positive control group (0.2mmol/L N-9) and 0.1mmol/L PD group spermicidal effect similar to that of 0.2mmol/L PD group of sperm is almost red fluorescence, spermicide the rate of 95%.
3.4 scanning electron microscopy and transmission electron microscopy in scanning electron microscope, the control group of normal sperm morphology and acrosome clearly, after treatment, showed a large number of sperm head and neck fracture under TEM. The control group, sperm cell membrane integrity, mitochondrial structural integrity. After treatment, the sperm membrane rupture, mitochondrial swelling, membrane or even fall off.
3.5Comet Assay experiment: negative control group, all experimental spermatozoa in group PD0.2mmol/L did not find DNA mobile tail, and the positive control group had obvious DNA mobile tail.
4. animal experiments
4.1 contraceptive effect: female SD rats were injected 0.9%NaCl100 l right uterus (control group), 3mg/ml PD100 L in the left uterine (dose of 0.3mg), and postoperative 2:1 male rats cage; second days after vaginal plug found cage, 10 days after the mice were killed, found that the injection of PD (left) side of the uterus no gestational sac, and saline injection side (right) normal uterine pregnancy.
4.2 vaginal stimulation test: female rats were randomly divided into 5 groups, every time to give 0.2ml (about 0.5g) PD gel vaginal administration, continuous administration for 14 days, after the last administration were animal, remove the vaginal tissue, fixed with formaldehyde for histological examination and vaginal tissue pathological stimuli score [40]. natural control blank gel group, 3mg/g group, PD group, 8mg/gPD group, 8mg/g group N-9 vaginal irritation scores were 2.11 + 2.01,3.22 + 1.07,4.89 + 0.69,7.44 + 1.58,3.67 + 1.53. vaginal irritation test score less than 8 points, irritation in the acceptable range:
PD instantly kill effect on human sperm, can cause sperm apoptosis, study the spermicidal mechanism showed that PD damage of sperm membrane. Spermicidally concentration of PD damage in DNA rats, PD in vivo experiments confirmed that the contraceptive effective contraception, the vaginal irritation in the acceptable range of.PD as a possible future the clinical potential of spermicide for contraception.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R169.4;R285

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