抗人尿激酶抗体对雌性小鼠生殖能力的影响
发布时间：2018-08-18 17:24
【摘要】： 第一部分诱导雌性小鼠产生抗人uPA抗体免疫方法和效价分析 目的建立一种利用人源uPA分子作为抗原诱导雌性昆明小鼠产生抗人uPA抗体的免疫方法,检测该免疫方法的有效性及稳定性。 方法将性成熟雌性昆明小鼠随机分为5组:空白对照组(不予任何处理)、佐剂对照组(100μl生理盐水+等体积佐剂)、低剂量免疫组(20μg/100μl uPA+等体积佐剂)、中剂量免疫组(40μg/100μl uPA+等体积佐剂)和高剂量免疫组(80μg/100μl uPA+等体积佐剂)。每2周皮下多点注射免疫1次,于第3次免疫后1周取其尾动脉血清以酶联免疫吸附实验(enzyme linked immunosorbent assay, ELISA)检测各组抗体效价,另外加设一组,即阴性对照组(PBS溶液代替样本血清)便于ELISA法检测中的比较。 结果以ELISA法检测各组血清样本中抗人uPA多克隆抗体相对含量,uPA三个剂量免疫组与三个对照组(阴性对照组、空白对照组和佐剂对照组)相比,抗人uPA多克隆抗体相对含量是增高的,差异具有统计学意义(P0.05);免疫组血清中抗人uPA多克隆抗体效价均达到1:10240以上。 结论利用人源uPA分子作为抗原诱导雌性昆明小鼠产生抗人uPA抗体的免疫方法,不仅可以建立一种使小鼠体内产生稳定uPA抗体的实验方法,而且小鼠体内的抗体可以达到较高水平。 第二部分抗人uPA抗体对雌性小鼠生殖能力的影响 实验一诱导雌性小鼠产生抗人uPA抗体后对其交配和妊娠情况的影响 目的采用人注射用uPA免疫雌性小鼠,小鼠体内产生抗人uPA抗体后,观察对雌性小鼠妊娠情况的影响。 方法动物分组及免疫方法同第一部分,于免疫4次后,分别将实验各组雌性小鼠与未经任何处理的同周龄雄性小鼠进行交配实验,于妊娠第7天解剖观察并记录妊娠率、平均胚胎着床数目。 结果uPA低、中、高剂量免疫组雌性小鼠的妊娠率以及胚胎着床数目与空白和佐剂对照组相比,妊娠率及胚胎着床数目降低,差异具有统计学意义(P0.05)。空白和佐剂对照组之间差异无统计学意义(P0.05)。 结论人注射用uPA免疫雌性小鼠,可干扰小鼠的生殖功能,使生育能力显著下降,提示uPA可能成为免疫避孕的有效靶点。 实验二诱导雌性小鼠产生抗人uPA抗体后对其体外受精能力的影响 目的研究采用人注射用uPA免疫雌性小鼠,小鼠体内产生抗人uPA抗体后,对小鼠的卵细胞体外受精能力影响,探讨uPA免疫使雌性小鼠生育力下降的机制。 方法动物分组及免疫方法同第一部分,于第12次免疫后1周,给予各实验组雌性小鼠PMSG/hCG促排卵后,获卵细胞团,记录卵细胞数目,与未经任何处理的健康雄性小鼠精子进行体外受精实验,记录受精率。卵巢组织经4%多聚甲醛固定,常规脱水、透明、包埋、切片、染色,记录黄体数目。 结果 1.排卵数目 uPA低、中、高剂量免疫组雌性小鼠排出的卵细胞数目分别与空白和佐剂对照组相比,明显减少,差异具有统计学意义(P0.05);空白和佐剂差异无统计学意义(P 0.05)。 2.体外受精率 与空白对照组相比,佐剂对照组、低、中、高剂量免疫组的受精率有所降低,但是差异无统计学意义(P0.05)。 3.卵裂情况 A+B级的卵裂的胚胎率在中、高剂量组占的比例明显比空白对照组、佐剂对照组和低剂量组要低,差异具有统计学意义(P0.01)。空白对照组、佐剂对照组及低剂量组之间差异无统计学意义(P 0.05)。 4.平均黄体数目 卵巢组织切片中平均黄体数目分别与空白和佐剂对照组相比,uPA低、中、高剂量免疫组明显减少,差异具有统计学意义(P0.05)。 结论采用人注射用uPA免疫雌性小鼠,小鼠体内产生抗人uPA抗体后,影响雌性小鼠卵泡排放、卵细胞受精能力,导致排卵数目、体外受精率和平均黄体数目下降,胚胎质量下降。 实验三体外实验观察含抗人uPA抗体血清干预正常雌性小鼠卵细胞对其受精能力的影响 目的观察该含抗人uPA抗体血清对正常雌性小鼠卵细胞的受精能力和存活情况的影响,探讨uPA免疫使雌性小鼠生育力下降的机制。 方法经过PMSG/hCG促排卵获得健康雌性小鼠卵细胞团,将该卵细胞团与各实验组的小鼠血清共同孵育30min后,再与未经任何处理的健康雄性小鼠精子进行体外受精实验,记录受精率和卵细胞死亡率。 结果 1.体外受精率 经过与uPA低、中、高剂量免疫组的血清共同孵育的卵细胞受精率比空白对照和佐剂对照组降低,并且差异有统计学意义(P0.01)。空白和佐剂对照组受精率比较,差异无统计学意义(P0.05)。 2.卵细胞的死亡率 含抗人uPA抗体的血清干预正常小鼠卵细胞,不仅受精率下降,并且明显的出现较多死亡的卵细胞,其中以高剂量免疫组卵细胞的死亡率为最高,高剂量组与空白组相比,差异有统计学意义(P 0.01),低、中剂量免疫组与空白和佐剂对照组相比,虽然死亡率有所升高,但是差异无统计学意义(P 0.05)。空白和佐剂对照组死亡率差异无统计学意义(P 0.05)。 结论以抗人uPA抗体血清干预正常小鼠卵细胞,观察该卵细胞体外受精情况,发现体外培养的卵细胞其体外受精率下降,死亡率升高,提示抗人uPA抗体血清可以降低正常雌性小鼠卵细胞的受精能力和卵细胞的存活能力。
[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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R96
本文编号：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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R96
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