小鼠子宫内膜干细胞的初步研究
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摘要
第一部分小鼠子宫标记滞留细胞的检测
目的:研究小鼠子宫内标记滞留细胞的存在及其分布特点。
方法:选择出生三天的雌性昆明白小乳鼠,实验组乳鼠皮下注射BrdU50ug/g,对照组皮下注射生理盐水100ul,每天2次,共注射3d。于最后一次注射后2h、1w、2w、4w和8w处死并取子宫固定,石蜡包埋用于免疫组化检测,每个时间段5只乳鼠。
结果:实验组标记后2h,小鼠腺体和基质中标记滞留细胞(LRCs)表达最高,分别为45.1%和57.4%,随着时间的增加,LRCs逐渐降低,到第8w在腺体中仅有极少量细胞表达,而基质细胞约有1.5%的LRCs,主要位于血管周围和内膜基层连接处。不同时相腺体和基质细胞中LRCs差异具有显著性(P<0.05)。
结论:昆明白小鼠子宫内LRCs主要位于血管周围和内膜基层连接处等,这些LRCs可能为子宫内膜干细胞。
第二部分体外培养小鼠子宫内膜形成克隆能力的检测
目的:通过体外培养检测小鼠子宫内膜细胞克隆能力。
方法:取出生3d的昆明白雌性小鼠,将其子宫剪碎后用0.125%胰酶消化,计数活细胞,按300个/cm2的密度接种到培养瓶中,培养12d左右。观察细胞及克隆生长特点,并测量细胞克隆大小和计算克隆形成率。同时取成年小鼠子宫培养作为对照。通过组织来源、细胞生长形态、细胞增殖能力以及角蛋白和波型蛋白免疫组化等进行鉴定,并用免疫荧光法检测腺体克隆Ki-67增殖指数。
结果:根据细胞组织来源分有腺体和基质两种克隆,根据细胞克隆大小和生长特点,分大克隆和小克隆两种类型。腺体大克隆和小克隆的大小分别为3.64±1.1和0.53±0.24mm,克隆形成率分别为0.051±0.013%和0.11±0.07%;基质大克隆和小克隆的直径大小分别为2.82±0.5和0.41±0.13mm,克隆形成率分别为0.008±0.001%和0.43±0.03%。四种克隆的克隆形成率均具有显著性差异(P<0.05),腺体和基质大克隆直径大小,腺体和基质小克隆直径大小分别两两比较,差异不均具有显著性(P>0.05),而腺体大小克隆大小比较,基质大小克隆分别两两比较,差异具有均显著性(P<0.05)。成年小鼠子宫有限稀释法原代培养无细胞克隆生长。腺体大克隆Ki-67增殖指数高于腺体小克隆(P<0.05)。
结论:子宫内膜细胞在体外培养中,可以形成单细胞克隆,腺体和基质细胞均可形成大克隆和小克隆。腺体大克隆增殖能力高于小克隆。
第三部分EGF和17β-雌二醇对细胞克隆生长的影响
目的:研究EGF和17β-雌二醇对子宫内膜细胞克隆生长的影响。
方法:在培养基中分别加入10ng/ml的EGF和5×10-8 mol/L的17β-雌二醇,按照第二部分方法对小鼠子宫进行原代培养,观察细胞形态,测量克隆大小并计算细胞克隆形成率,同时用无特殊添加成分的培养基培养作为对照。
结果:与对照组比较,EGF组四种克隆形成率且克隆直径均值略高,但是差异不具有显著性(均P>0.05)。17β-雌二醇组两种小克隆克隆形成率均值略高,而两种大克隆克隆形成率较低,与对照组分别比较差异不具有显著性(均P>0.05)。17β-雌二醇组腺体大克隆大于对照组,差异具有显著性(P<0.05), 17β-雌二醇组与EGF组比较,细胞克隆形成率和大小差异均不具有显著性(P>0.05)。
结论:添加EGF和17β-雌二醇对小鼠子宫在体外培养时,并不能增加其克隆形成率,雌激素在体外有促进腺体克隆细胞分化的作用。
第四部分小鼠间充质干细胞向子宫内膜方向分化的实验研究
目的:探索小鼠骨髓间充质干细胞向子宫内膜的迁移和分化
方法: 1.体外原代培养雄性小鼠骨髓间充质干细胞,并在体外用BrdU体外标记,用CD34和CD29抗体进行免疫组化鉴定,用BrdU抗体检测其标记阳性率。
2.骨髓移植模型:雌性小鼠放疗后,从鼠尾静脉注射移植间充质干细胞,作为放疗后移植组;同时设放疗对照和正常对照组。每组8只小鼠。
3.观察小鼠的一般情况,在小鼠濒死前或者生存>60d后,处死小鼠,取其子宫一侧固定石蜡包埋,用于BrdU免疫组化;另一侧子宫提取DNA,用于PCR检测Sry基因表达。
结果: 1.间充质干细胞呈梭形或多角形,经免疫组化鉴定CD29(+)和CD34(-),符合间充质干细胞特性。Brdu标记阳性率约为80%。
2.骨髓移植模型:放疗对照组在放疗后14d小鼠死亡7/8只;移植组移植后4只存活时间<20d,2只存活时间>30d,2只存活时间>60d。放疗对照组、正常对照组和移植组早期死亡小鼠子宫Brdu检测为阴性。移植组短期存活和长期存活的小鼠,Brdu阳性主要表达在子宫腺体和基质之间的毛细血管内皮细胞及少量基质细胞核内。正常对照组和放疗组小鼠子宫SRY基因检测为阴性。移植组早期死亡、短期存活和长期存活的小鼠子宫SRY基因表达均为阳性。
结论:小鼠间充质干细胞可以迁移到子宫,并有可能成为子宫内膜修复的细胞来源。
Part I The existence of endometrial stem cell in mouse uterus
Objective:
To study the existence of endometrial stem cell and its distribution in mouse uterus．
Methods:
Selected female Kunming white mice after born three days, subcutaneous injected BrdU50ug/g in experiment group and 0.9%NS in control group twice daily andtotally injected 3 d. Put mice death after injected 2h、1w、2w、4w和8w, fixed the uterus forimmunohistochemistry and pathology, 5 mice for each phase.
Results:
The expression of LRCs in stroma and gland cells after 2h were 45.1% and 57.4%respectively and which were gradually degraded. Only few LRCs in the gland bottom in 8wand 1.5% LRCs in stroma cells mainly located around the blood vessel andendometrial-myometrial junction. LRCs in different phases of stroma and gland cells weresignificance（P<0.05）.
Conclusions:
There were endometrial stem cells in the uterus of Kunming white mice and mainly locatedaround the blood vessel and endometrial-myometrial junction.
Part II The cloning efficiency of mouse endometrium in vitro primaryculture
Objective:
To study the cloning efficiency of mouse endometrium in vitro primary culture by limiteddilution technology.
Methods:
Selected the female kuming mice after born 3 days, sheared and digested the uterus by0.125% pancreatic enzyme, counting the living cells and inoculating them to culture flask.Observed the characteristics of cell clones during 12days and measured the sizes of clonesand calculated the rate of cloning efficiency. At the same time, cultured the uterus cells ofadult female mice as control. Identification the category of the cells by keratin and vimentinby immunohistochemistry and appearance of the cells、the origin of tissue. Detected theproliferation index of Ki-67 in gland clones by immunofluorescence.
Results:
There were stroma and gland clones divided by the cell origin, large and little clonesdivided by size and growth characteristics. The sizes of large and little gland clones wererespectively 3.64±1.1 and 0.53±0.24mm, the cloning efficiency was respectively 0.051%±0.013 and 0.11±0.07%；The sizes of large and little stroma clones was respectively 2.82±0.5 and 0.41±0.13 mm, the cloning efficiency was respectively 0.008±0.001% and0.43±0.03%. There was no cell growth in the adult uterus by limited dilution technology.The difference of the cloning efficiency rates of four kids of clones were all significant（P<0.05）.Compared the sizes of two kinds of large clones or the two kinds of little clones,the differences were not significan（t P＞0.05）, The PI of Ki-67 was higher in the nucleus oflarge clones.
Conclusion:
The endometrium cells can form monocell clones in vitro primary culture which includedlarge and little clones. The proliferation efficiency of the large gland clone was higher.
Part III The effects of EGF and 17β-Estradiol to the growth of cellclones
Objective:
To study the effects of EGF and 17β-Estradiol on the growth of cell clones.
Methods:
Added 10ng/ml EGF or 5×10-8 mol/L 17β-Estradiol into the medium, cultured the cell asthe methods of the part II and observed the cell appearance、measured the sizes of clonesand calculated the rate of cloning efficiency.
Results:
The rates of cloning efficiency in the EGF groups were higher than the control group, thedifference were not significant（P>0.05）. The cloning efficiency rates of little clones in17β-Estradiol groups were higher than the control group, while the cloning efficiency ratesof large clones were lower, the difference were not significan（t P>0.05）. The large clones in17β-Estradiol groups were larger than the control group and the difference were significant（P<0.05）.The difference of the cloning efficiency rates and the sizes of the clones werenot significance in the EGF groups and 17β-Estradiol groups（P>0.05）.
Conclutions:
when added 10ng/ml EGF or 5×10-8 mol/L 17β-Estradiol into the medium, the rates ofcloning efficiency were not higher. 17β-Estradiol could promote the differentiation of thegland clones.
Part IV The experiment research of mesenchymal stem cellsdifferentiation to the endometrium
Objective:
To study the immigration and differentiation of mesenchymal stem cells to endometrium.
Methods:
1. Cultured the male MSCs and labeling the Brdu in vitro, then identified by CD34 andCD29; the positive rate of Brdu was examined by immunohistochemistry.
2. After radiotherapy, the MSCs were injected through the tail vein of female mouse astransplantation group. The other groups were radiotherapy group and normal control group.Each groups had 8 mice.
3. Observe the general condition of mice and put them death before died or living longer>60d, taken one uterus for immunohistochemistry and pathology the others for PCR and.
Results:
1.The MSCs was CD29（+）and CD34（-）and the Brdu positive rate was roughly 80%.
2. 7/8 mice died in 14 days; 4 mice lived <20d; 2 mice survived longer >30d; 2 mice livedlonger >60d in the radiotherapy group. The Brdu was negative in radiotherapy、normalcontrol groups and early died mice in experimental group. In the long-living andshort-living mice of transplantation group, Brdu expressed on the endothelial cells of bloodcapillary and few stroma cells. The SRY gene was negative in radiotherapy、normal controlgroups and early died mice in experimental group, but it was positive in the uterus oflong-living and short-living mice.
Conclusion:
The mouse MSCs can immgrate to the uterus and maybe one of the origin ofendometrium.
目的:研究小鼠子宫内标记滞留细胞的存在及其分布特点。
方法:选择出生三天的雌性昆明白小乳鼠,实验组乳鼠皮下注射BrdU50ug/g,对照组皮下注射生理盐水100ul,每天2次,共注射3d。于最后一次注射后2h、1w、2w、4w和8w处死并取子宫固定,石蜡包埋用于免疫组化检测,每个时间段5只乳鼠。
结果:实验组标记后2h,小鼠腺体和基质中标记滞留细胞(LRCs)表达最高,分别为45.1%和57.4%,随着时间的增加,LRCs逐渐降低,到第8w在腺体中仅有极少量细胞表达,而基质细胞约有1.5%的LRCs,主要位于血管周围和内膜基层连接处。不同时相腺体和基质细胞中LRCs差异具有显著性(P<0.05)。
结论:昆明白小鼠子宫内LRCs主要位于血管周围和内膜基层连接处等,这些LRCs可能为子宫内膜干细胞。
第二部分体外培养小鼠子宫内膜形成克隆能力的检测
目的:通过体外培养检测小鼠子宫内膜细胞克隆能力。
方法:取出生3d的昆明白雌性小鼠,将其子宫剪碎后用0.125%胰酶消化,计数活细胞,按300个/cm2的密度接种到培养瓶中,培养12d左右。观察细胞及克隆生长特点,并测量细胞克隆大小和计算克隆形成率。同时取成年小鼠子宫培养作为对照。通过组织来源、细胞生长形态、细胞增殖能力以及角蛋白和波型蛋白免疫组化等进行鉴定,并用免疫荧光法检测腺体克隆Ki-67增殖指数。
结果:根据细胞组织来源分有腺体和基质两种克隆,根据细胞克隆大小和生长特点,分大克隆和小克隆两种类型。腺体大克隆和小克隆的大小分别为3.64±1.1和0.53±0.24mm,克隆形成率分别为0.051±0.013%和0.11±0.07%;基质大克隆和小克隆的直径大小分别为2.82±0.5和0.41±0.13mm,克隆形成率分别为0.008±0.001%和0.43±0.03%。四种克隆的克隆形成率均具有显著性差异(P<0.05),腺体和基质大克隆直径大小,腺体和基质小克隆直径大小分别两两比较,差异不均具有显著性(P>0.05),而腺体大小克隆大小比较,基质大小克隆分别两两比较,差异具有均显著性(P<0.05)。成年小鼠子宫有限稀释法原代培养无细胞克隆生长。腺体大克隆Ki-67增殖指数高于腺体小克隆(P<0.05)。
结论:子宫内膜细胞在体外培养中,可以形成单细胞克隆,腺体和基质细胞均可形成大克隆和小克隆。腺体大克隆增殖能力高于小克隆。
第三部分EGF和17β-雌二醇对细胞克隆生长的影响
目的:研究EGF和17β-雌二醇对子宫内膜细胞克隆生长的影响。
方法:在培养基中分别加入10ng/ml的EGF和5×10-8 mol/L的17β-雌二醇,按照第二部分方法对小鼠子宫进行原代培养,观察细胞形态,测量克隆大小并计算细胞克隆形成率,同时用无特殊添加成分的培养基培养作为对照。
结果:与对照组比较,EGF组四种克隆形成率且克隆直径均值略高,但是差异不具有显著性(均P>0.05)。17β-雌二醇组两种小克隆克隆形成率均值略高,而两种大克隆克隆形成率较低,与对照组分别比较差异不具有显著性(均P>0.05)。17β-雌二醇组腺体大克隆大于对照组,差异具有显著性(P<0.05), 17β-雌二醇组与EGF组比较,细胞克隆形成率和大小差异均不具有显著性(P>0.05)。
结论:添加EGF和17β-雌二醇对小鼠子宫在体外培养时,并不能增加其克隆形成率,雌激素在体外有促进腺体克隆细胞分化的作用。
第四部分小鼠间充质干细胞向子宫内膜方向分化的实验研究
目的:探索小鼠骨髓间充质干细胞向子宫内膜的迁移和分化
方法: 1.体外原代培养雄性小鼠骨髓间充质干细胞,并在体外用BrdU体外标记,用CD34和CD29抗体进行免疫组化鉴定,用BrdU抗体检测其标记阳性率。
2.骨髓移植模型:雌性小鼠放疗后,从鼠尾静脉注射移植间充质干细胞,作为放疗后移植组;同时设放疗对照和正常对照组。每组8只小鼠。
3.观察小鼠的一般情况,在小鼠濒死前或者生存>60d后,处死小鼠,取其子宫一侧固定石蜡包埋,用于BrdU免疫组化;另一侧子宫提取DNA,用于PCR检测Sry基因表达。
结果: 1.间充质干细胞呈梭形或多角形,经免疫组化鉴定CD29(+)和CD34(-),符合间充质干细胞特性。Brdu标记阳性率约为80%。
2.骨髓移植模型:放疗对照组在放疗后14d小鼠死亡7/8只;移植组移植后4只存活时间<20d,2只存活时间>30d,2只存活时间>60d。放疗对照组、正常对照组和移植组早期死亡小鼠子宫Brdu检测为阴性。移植组短期存活和长期存活的小鼠,Brdu阳性主要表达在子宫腺体和基质之间的毛细血管内皮细胞及少量基质细胞核内。正常对照组和放疗组小鼠子宫SRY基因检测为阴性。移植组早期死亡、短期存活和长期存活的小鼠子宫SRY基因表达均为阳性。
结论:小鼠间充质干细胞可以迁移到子宫,并有可能成为子宫内膜修复的细胞来源。
Part I The existence of endometrial stem cell in mouse uterus
Objective:
To study the existence of endometrial stem cell and its distribution in mouse uterus．
Methods:
Selected female Kunming white mice after born three days, subcutaneous injected BrdU50ug/g in experiment group and 0.9%NS in control group twice daily andtotally injected 3 d. Put mice death after injected 2h、1w、2w、4w和8w, fixed the uterus forimmunohistochemistry and pathology, 5 mice for each phase.
Results:
The expression of LRCs in stroma and gland cells after 2h were 45.1% and 57.4%respectively and which were gradually degraded. Only few LRCs in the gland bottom in 8wand 1.5% LRCs in stroma cells mainly located around the blood vessel andendometrial-myometrial junction. LRCs in different phases of stroma and gland cells weresignificance（P<0.05）.
Conclusions:
There were endometrial stem cells in the uterus of Kunming white mice and mainly locatedaround the blood vessel and endometrial-myometrial junction.
Part II The cloning efficiency of mouse endometrium in vitro primaryculture
Objective:
To study the cloning efficiency of mouse endometrium in vitro primary culture by limiteddilution technology.
Methods:
Selected the female kuming mice after born 3 days, sheared and digested the uterus by0.125% pancreatic enzyme, counting the living cells and inoculating them to culture flask.Observed the characteristics of cell clones during 12days and measured the sizes of clonesand calculated the rate of cloning efficiency. At the same time, cultured the uterus cells ofadult female mice as control. Identification the category of the cells by keratin and vimentinby immunohistochemistry and appearance of the cells、the origin of tissue. Detected theproliferation index of Ki-67 in gland clones by immunofluorescence.
Results:
There were stroma and gland clones divided by the cell origin, large and little clonesdivided by size and growth characteristics. The sizes of large and little gland clones wererespectively 3.64±1.1 and 0.53±0.24mm, the cloning efficiency was respectively 0.051%±0.013 and 0.11±0.07%；The sizes of large and little stroma clones was respectively 2.82±0.5 and 0.41±0.13 mm, the cloning efficiency was respectively 0.008±0.001% and0.43±0.03%. There was no cell growth in the adult uterus by limited dilution technology.The difference of the cloning efficiency rates of four kids of clones were all significant（P<0.05）.Compared the sizes of two kinds of large clones or the two kinds of little clones,the differences were not significan（t P＞0.05）, The PI of Ki-67 was higher in the nucleus oflarge clones.
Conclusion:
The endometrium cells can form monocell clones in vitro primary culture which includedlarge and little clones. The proliferation efficiency of the large gland clone was higher.
Part III The effects of EGF and 17β-Estradiol to the growth of cellclones
Objective:
To study the effects of EGF and 17β-Estradiol on the growth of cell clones.
Methods:
Added 10ng/ml EGF or 5×10-8 mol/L 17β-Estradiol into the medium, cultured the cell asthe methods of the part II and observed the cell appearance、measured the sizes of clonesand calculated the rate of cloning efficiency.
Results:
The rates of cloning efficiency in the EGF groups were higher than the control group, thedifference were not significant（P>0.05）. The cloning efficiency rates of little clones in17β-Estradiol groups were higher than the control group, while the cloning efficiency ratesof large clones were lower, the difference were not significan（t P>0.05）. The large clones in17β-Estradiol groups were larger than the control group and the difference were significant（P<0.05）.The difference of the cloning efficiency rates and the sizes of the clones werenot significance in the EGF groups and 17β-Estradiol groups（P>0.05）.
Conclutions:
when added 10ng/ml EGF or 5×10-8 mol/L 17β-Estradiol into the medium, the rates ofcloning efficiency were not higher. 17β-Estradiol could promote the differentiation of thegland clones.
Part IV The experiment research of mesenchymal stem cellsdifferentiation to the endometrium
Objective:
To study the immigration and differentiation of mesenchymal stem cells to endometrium.
Methods:
1. Cultured the male MSCs and labeling the Brdu in vitro, then identified by CD34 andCD29; the positive rate of Brdu was examined by immunohistochemistry.
2. After radiotherapy, the MSCs were injected through the tail vein of female mouse astransplantation group. The other groups were radiotherapy group and normal control group.Each groups had 8 mice.
3. Observe the general condition of mice and put them death before died or living longer>60d, taken one uterus for immunohistochemistry and pathology the others for PCR and.
Results:
1.The MSCs was CD29（+）and CD34（-）and the Brdu positive rate was roughly 80%.
2. 7/8 mice died in 14 days; 4 mice lived <20d; 2 mice survived longer >30d; 2 mice livedlonger >60d in the radiotherapy group. The Brdu was negative in radiotherapy、normalcontrol groups and early died mice in experimental group. In the long-living andshort-living mice of transplantation group, Brdu expressed on the endothelial cells of bloodcapillary and few stroma cells. The SRY gene was negative in radiotherapy、normal controlgroups and early died mice in experimental group, but it was positive in the uterus oflong-living and short-living mice.
Conclusion:
The mouse MSCs can immgrate to the uterus and maybe one of the origin ofendometrium.
引文
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