抗人尿激酶抗體對雌性小鼠生殖能力的影響
發(fā)布時間：2018-08-18 17:24
【摘要】： 第一部分誘導(dǎo)雌性小鼠產(chǎn)生抗人uPA抗體免疫方法和效價(jià)分析 目的建立一種利用人源uPA分子作為抗原誘導(dǎo)雌性昆明小鼠產(chǎn)生抗人uPA抗體的免疫方法,檢測該免疫方法的有效性及穩(wěn)定性。 方法將性成熟雌性昆明小鼠隨機(jī)分為5組:空白對照組(不予任何處理)、佐劑對照組(100μl生理鹽水+等體積佐劑)、低劑量免疫組(20μg/100μl uPA+等體積佐劑)、中劑量免疫組(40μg/100μl uPA+等體積佐劑)和高劑量免疫組(80μg/100μl uPA+等體積佐劑)。每2周皮下多點(diǎn)注射免疫1次,于第3次免疫后1周取其尾動脈血清以酶聯(lián)免疫吸附實(shí)驗(yàn)(enzyme linked immunosorbent assay, ELISA)檢測各組抗體效價(jià),另外加設(shè)一組,即陰性對照組(PBS溶液代替樣本血清)便于ELISA法檢測中的比較。 結(jié)果以ELISA法檢測各組血清樣本中抗人uPA多克隆抗體相對含量,uPA三個劑量免疫組與三個對照組(陰性對照組、空白對照組和佐劑對照組)相比,抗人uPA多克隆抗體相對含量是增高的,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);免疫組血清中抗人uPA多克隆抗體效價(jià)均達(dá)到1:10240以上。 結(jié)論利用人源uPA分子作為抗原誘導(dǎo)雌性昆明小鼠產(chǎn)生抗人uPA抗體的免疫方法,不僅可以建立一種使小鼠體內(nèi)產(chǎn)生穩(wěn)定uPA抗體的實(shí)驗(yàn)方法,而且小鼠體內(nèi)的抗體可以達(dá)到較高水平。 第二部分抗人uPA抗體對雌性小鼠生殖能力的影響 實(shí)驗(yàn)一誘導(dǎo)雌性小鼠產(chǎn)生抗人uPA抗體后對其交配和妊娠情況的影響 目的采用人注射用uPA免疫雌性小鼠,小鼠體內(nèi)產(chǎn)生抗人uPA抗體后,觀察對雌性小鼠妊娠情況的影響。 方法動物分組及免疫方法同第一部分,于免疫4次后,分別將實(shí)驗(yàn)各組雌性小鼠與未經(jīng)任何處理的同周齡雄性小鼠進(jìn)行交配實(shí)驗(yàn),于妊娠第7天解剖觀察并記錄妊娠率、平均胚胎著床數(shù)目。 結(jié)果uPA低、中、高劑量免疫組雌性小鼠的妊娠率以及胚胎著床數(shù)目與空白和佐劑對照組相比,妊娠率及胚胎著床數(shù)目降低,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)�？瞻缀妥魟⿲φ战M之間差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論人注射用uPA免疫雌性小鼠,可干擾小鼠的生殖功能,使生育能力顯著下降,提示uPA可能成為免疫避孕的有效靶點(diǎn)。 實(shí)驗(yàn)二誘導(dǎo)雌性小鼠產(chǎn)生抗人uPA抗體后對其體外受精能力的影響 目的研究采用人注射用uPA免疫雌性小鼠,小鼠體內(nèi)產(chǎn)生抗人uPA抗體后,對小鼠的卵細(xì)胞體外受精能力影響,探討uPA免疫使雌性小鼠生育力下降的機(jī)制。 方法動物分組及免疫方法同第一部分,于第12次免疫后1周,給予各實(shí)驗(yàn)組雌性小鼠PMSG/hCG促排卵后,獲卵細(xì)胞團(tuán),記錄卵細(xì)胞數(shù)目,與未經(jīng)任何處理的健康雄性小鼠精子進(jìn)行體外受精實(shí)驗(yàn),記錄受精率。卵巢組織經(jīng)4%多聚甲醛固定,常規(guī)脫水、透明、包埋、切片、染色,記錄黃體數(shù)目。 結(jié)果 1.排卵數(shù)目 uPA低、中、高劑量免疫組雌性小鼠排出的卵細(xì)胞數(shù)目分別與空白和佐劑對照組相比,明顯減少,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);空白和佐劑差異無統(tǒng)計(jì)學(xué)意義(P 0.05)。 2.體外受精率 與空白對照組相比,佐劑對照組、低、中、高劑量免疫組的受精率有所降低,但是差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 3.卵裂情況 A+B級的卵裂的胚胎率在中、高劑量組占的比例明顯比空白對照組、佐劑對照組和低劑量組要低,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。空白對照組、佐劑對照組及低劑量組之間差異無統(tǒng)計(jì)學(xué)意義(P 0.05)。 4.平均黃體數(shù)目 卵巢組織切片中平均黃體數(shù)目分別與空白和佐劑對照組相比,uPA低、中、高劑量免疫組明顯減少,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論采用人注射用uPA免疫雌性小鼠,小鼠體內(nèi)產(chǎn)生抗人uPA抗體后,影響雌性小鼠卵泡排放、卵細(xì)胞受精能力,導(dǎo)致排卵數(shù)目、體外受精率和平均黃體數(shù)目下降,胚胎質(zhì)量下降。 實(shí)驗(yàn)三體外實(shí)驗(yàn)觀察含抗人uPA抗體血清干預(yù)正常雌性小鼠卵細(xì)胞對其受精能力的影響 目的觀察該含抗人uPA抗體血清對正常雌性小鼠卵細(xì)胞的受精能力和存活情況的影響,探討uPA免疫使雌性小鼠生育力下降的機(jī)制。 方法經(jīng)過PMSG/hCG促排卵獲得健康雌性小鼠卵細(xì)胞團(tuán),將該卵細(xì)胞團(tuán)與各實(shí)驗(yàn)組的小鼠血清共同孵育30min后,再與未經(jīng)任何處理的健康雄性小鼠精子進(jìn)行體外受精實(shí)驗(yàn),記錄受精率和卵細(xì)胞死亡率。 結(jié)果 1.體外受精率 經(jīng)過與uPA低、中、高劑量免疫組的血清共同孵育的卵細(xì)胞受精率比空白對照和佐劑對照組降低,并且差異有統(tǒng)計(jì)學(xué)意義(P0.01)�？瞻缀妥魟⿲φ战M受精率比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 2.卵細(xì)胞的死亡率 含抗人uPA抗體的血清干預(yù)正常小鼠卵細(xì)胞,不僅受精率下降,并且明顯的出現(xiàn)較多死亡的卵細(xì)胞,其中以高劑量免疫組卵細(xì)胞的死亡率為最高,高劑量組與空白組相比,差異有統(tǒng)計(jì)學(xué)意義(P 0.01),低、中劑量免疫組與空白和佐劑對照組相比,雖然死亡率有所升高,但是差異無統(tǒng)計(jì)學(xué)意義(P 0.05)。空白和佐劑對照組死亡率差異無統(tǒng)計(jì)學(xué)意義(P 0.05)。 結(jié)論以抗人uPA抗體血清干預(yù)正常小鼠卵細(xì)胞,觀察該卵細(xì)胞體外受精情況,發(fā)現(xiàn)體外培養(yǎng)的卵細(xì)胞其體外受精率下降,死亡率升高,提示抗人uPA抗體血清可以降低正常雌性小鼠卵細(xì)胞的受精能力和卵細(xì)胞的存活能力。
[Abstract]:Part one immunization method and titer analysis of inducing female mice to produce anti human uPA antibody
Objective To establish an immunoassay for the production of anti-human uPA antibody in female Kunming mice induced by human uPA molecule as antigen, and to test its effectiveness and stability.
Methods Sexually mature female Kunming mice were randomly divided into five groups: blank control group (without any treatment), adjuvant control group (100 ml normal saline + equal volume adjuvant), low dose immunization group (20 UG / 100 UG uPA + equal volume adjuvant), medium dose immunization group (40 UG / 100 UG uPA + equal volume adjuvant) and high dose immunization group (80 UG / 100 UG uPA + equal volume adjuvant). The tail artery serum was taken one week after the third immunization. The titers of antibodies in each group were detected by enzyme linked immunosorbent assay (ELISA). Another group, negative control group (PBS solution instead of sample serum), was added to facilitate the comparison of ELISA.
Results The relative content of anti-human uPA polyclonal antibody in serum samples of each group was detected by ELISA. Compared with three control groups (negative control group, blank control group and adjuvant control group), the relative content of anti-human uPA polyclonal antibody in serum of immune group increased significantly (P 0.05). The titer of antibody reached over 1:10240.
Conclusion Using human uPA molecule as antigen to induce female Kunming mice to produce anti-human uPA antibody can not only establish an experimental method to produce stable uPA antibody in mice, but also achieve a higher level of antibody in mice.
The effect of second anti human uPA antibody on reproductive ability of female mice
Experiment 1 induced the female mice to produce anti human uPA antibodies and their effects on mating and pregnancy.
Objective To observe the effect of human uPA injection on pregnancy in female mice after immunizing female mice with anti-human uPA antibody.
Methods Animal grouping and immunization methods were the same as those in the first part. After 4 times of immunization, the female mice in each group were mated with male mice of the same age without any treatment. The pregnancy rate and the average number of embryo implantation were observed and recorded on the 7th day of pregnancy.
Results Compared with the blank and adjuvant control group, the pregnancy rate and the number of embryo implantation in the low, medium and high dose uPA group were significantly lower (P 0.05). There was no significant difference between the blank and adjuvant control group (P 0.05).
Conclusion Immunization of female mice with human uPA for injection can interfere with the reproductive function of mice and decrease their fertility significantly, suggesting that uPA may be an effective target for immunocontraception.
Effect of experiment two on the in vitro fertilization ability of female mice induced by anti human uPA antibody
Objective To study the effect of human uPA on in vitro fertilization of mouse oocytes and the mechanism of fertility decline in female mice immunized with uPA.
Methods Animal grouping and immunization methods were the same as those in the first part. One week after the 12th immunization, female mice in each experimental group were given PMSG/hCG for ovulation induction. Oocyte mass was obtained and the number of oocytes was recorded. The fertilization rate was recorded in vitro with sperm of healthy male mice without any treatment. The water was transparent, embedded, sliced, stained, and the number of corpus luteum was recorded.
Result
1. number of ovulation
The number of oocytes excreted by female mice with low, medium and high dose of uPA was significantly lower than that of blank and adjuvant control groups (P 0.05), and there was no significant difference between blank and adjuvant groups (P 0.05).
2. in vitro fertilization rate
Compared with the blank control group, the fertilization rate of adjuvant control group, low, medium and high dose immunization group decreased, but the difference was not statistically significant (P 0.05).
3. cleavage
The embryo rate of A+B cleavage was significantly lower in the high dose group than in the blank control group, the adjuvant control group and the low dose group (P 0.01). There was no significant difference between the blank control group, the adjuvant control group and the low dose group (P 0.05).
4. the average number of corpus luteum
The average number of corpus luteum in ovarian tissue slices was significantly lower in uPA group than in control group and adjuvant group (P 0.05).
Conclusion Female mice were immunized with human uPA for injection. The production of anti-human uPA antibody in vivo affected the follicular discharge and fertilization ability of female mice, resulting in the decrease of ovulation number, in vitro fertilization rate and average luteal number, and the decrease of embryo quality.
Effect of serum containing anti-human uPA antibody on fertilization of normal female mouse oocytes in vitro
Objective To observe the effect of the serum containing anti-human uPA antibody on the fertilization and survival of normal female mice eggs, and to explore the mechanism of fertility decline in female mice induced by uPA immunization.
Methods The oocyte mass of healthy female mice was obtained by PMSG/hCG ovulation induction. The oocyte mass was incubated with mouse serum of each experimental group for 30 minutes, and then fertilized in vitro with sperm of healthy male mice without any treatment. The fertilization rate and oocyte death rate were recorded.
Result
1. in vitro fertilization rate
The fertilization rate of oocytes co-incubated with low, medium and high dose of uPA was lower than that of the control group and the adjuvant group, and the difference was statistically significant (P 0.01). There was no significant difference between the blank group and the adjuvant group (P 0.05).
2. death rate of eggs
The serum containing anti-human uPA antibody interfered with normal mouse oocytes, not only fertilization rate decreased, but also more dead oocytes appeared obviously. The mortality of oocytes in the high-dose immunization group was the highest, and the mortality of oocytes in the high-dose immunization group was significantly higher than that in the blank group (P 0.01). There was no significant difference in mortality between the control group and the blank group (P 0.05).
Conclusion Anti-human uPA antibody serum can interfere with normal mouse oocytes and observe the in vitro fertilization of the oocytes. It is found that the in vitro fertilization rate of the oocytes cultured in vitro decreases and the mortality rate increases, suggesting that anti-human uPA antibody serum can reduce the fertilization ability and the viability of the oocytes of normal female mice.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392;R96
本文編號：2190160
[Abstract]:Part one immunization method and titer analysis of inducing female mice to produce anti human uPA antibody
Objective To establish an immunoassay for the production of anti-human uPA antibody in female Kunming mice induced by human uPA molecule as antigen, and to test its effectiveness and stability.
Methods Sexually mature female Kunming mice were randomly divided into five groups: blank control group (without any treatment), adjuvant control group (100 ml normal saline + equal volume adjuvant), low dose immunization group (20 UG / 100 UG uPA + equal volume adjuvant), medium dose immunization group (40 UG / 100 UG uPA + equal volume adjuvant) and high dose immunization group (80 UG / 100 UG uPA + equal volume adjuvant). The tail artery serum was taken one week after the third immunization. The titers of antibodies in each group were detected by enzyme linked immunosorbent assay (ELISA). Another group, negative control group (PBS solution instead of sample serum), was added to facilitate the comparison of ELISA.
Results The relative content of anti-human uPA polyclonal antibody in serum samples of each group was detected by ELISA. Compared with three control groups (negative control group, blank control group and adjuvant control group), the relative content of anti-human uPA polyclonal antibody in serum of immune group increased significantly (P 0.05). The titer of antibody reached over 1:10240.
Conclusion Using human uPA molecule as antigen to induce female Kunming mice to produce anti-human uPA antibody can not only establish an experimental method to produce stable uPA antibody in mice, but also achieve a higher level of antibody in mice.
The effect of second anti human uPA antibody on reproductive ability of female mice
Experiment 1 induced the female mice to produce anti human uPA antibodies and their effects on mating and pregnancy.
Objective To observe the effect of human uPA injection on pregnancy in female mice after immunizing female mice with anti-human uPA antibody.
Methods Animal grouping and immunization methods were the same as those in the first part. After 4 times of immunization, the female mice in each group were mated with male mice of the same age without any treatment. The pregnancy rate and the average number of embryo implantation were observed and recorded on the 7th day of pregnancy.
Results Compared with the blank and adjuvant control group, the pregnancy rate and the number of embryo implantation in the low, medium and high dose uPA group were significantly lower (P 0.05). There was no significant difference between the blank and adjuvant control group (P 0.05).
Conclusion Immunization of female mice with human uPA for injection can interfere with the reproductive function of mice and decrease their fertility significantly, suggesting that uPA may be an effective target for immunocontraception.
Effect of experiment two on the in vitro fertilization ability of female mice induced by anti human uPA antibody
Objective To study the effect of human uPA on in vitro fertilization of mouse oocytes and the mechanism of fertility decline in female mice immunized with uPA.
Methods Animal grouping and immunization methods were the same as those in the first part. One week after the 12th immunization, female mice in each experimental group were given PMSG/hCG for ovulation induction. Oocyte mass was obtained and the number of oocytes was recorded. The fertilization rate was recorded in vitro with sperm of healthy male mice without any treatment. The water was transparent, embedded, sliced, stained, and the number of corpus luteum was recorded.
Result
1. number of ovulation
The number of oocytes excreted by female mice with low, medium and high dose of uPA was significantly lower than that of blank and adjuvant control groups (P 0.05), and there was no significant difference between blank and adjuvant groups (P 0.05).
2. in vitro fertilization rate
Compared with the blank control group, the fertilization rate of adjuvant control group, low, medium and high dose immunization group decreased, but the difference was not statistically significant (P 0.05).
3. cleavage
The embryo rate of A+B cleavage was significantly lower in the high dose group than in the blank control group, the adjuvant control group and the low dose group (P 0.01). There was no significant difference between the blank control group, the adjuvant control group and the low dose group (P 0.05).
4. the average number of corpus luteum
The average number of corpus luteum in ovarian tissue slices was significantly lower in uPA group than in control group and adjuvant group (P 0.05).
Conclusion Female mice were immunized with human uPA for injection. The production of anti-human uPA antibody in vivo affected the follicular discharge and fertilization ability of female mice, resulting in the decrease of ovulation number, in vitro fertilization rate and average luteal number, and the decrease of embryo quality.
Effect of serum containing anti-human uPA antibody on fertilization of normal female mouse oocytes in vitro
Objective To observe the effect of the serum containing anti-human uPA antibody on the fertilization and survival of normal female mice eggs, and to explore the mechanism of fertility decline in female mice induced by uPA immunization.
Methods The oocyte mass of healthy female mice was obtained by PMSG/hCG ovulation induction. The oocyte mass was incubated with mouse serum of each experimental group for 30 minutes, and then fertilized in vitro with sperm of healthy male mice without any treatment. The fertilization rate and oocyte death rate were recorded.
Result
1. in vitro fertilization rate
The fertilization rate of oocytes co-incubated with low, medium and high dose of uPA was lower than that of the control group and the adjuvant group, and the difference was statistically significant (P 0.01). There was no significant difference between the blank group and the adjuvant group (P 0.05).
2. death rate of eggs
The serum containing anti-human uPA antibody interfered with normal mouse oocytes, not only fertilization rate decreased, but also more dead oocytes appeared obviously. The mortality of oocytes in the high-dose immunization group was the highest, and the mortality of oocytes in the high-dose immunization group was significantly higher than that in the blank group (P 0.01). There was no significant difference in mortality between the control group and the blank group (P 0.05).
Conclusion Anti-human uPA antibody serum can interfere with normal mouse oocytes and observe the in vitro fertilization of the oocytes. It is found that the in vitro fertilization rate of the oocytes cultured in vitro decreases and the mortality rate increases, suggesting that anti-human uPA antibody serum can reduce the fertilization ability and the viability of the oocytes of normal female mice.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392;R96
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