摘要
目的：
探索全胚和胎盘组织细胞悬液及其间充质干细胞的抗衰老作用。
方法：
1.小鼠胎鼠、胎盘组织细胞悬液的制备以及小鼠胎鼠、胎盘源间充质干细胞的分离、培养和鉴定
剖宫产取6日龄BALB/c小鼠胎鼠及孕18天BALB/c小鼠胎盘(皆雌雄混合),通过吹打悬浮、研磨、过滤等机械办法制备小鼠胎鼠、胎盘组织细胞悬液；通过密度梯度离心法、两步离心法结合贴壁培养法,从小鼠胎鼠、胎盘组织中获取间充质干细胞,鉴定细胞表面标志；诱导细胞向脂肪细胞、成骨细胞分化,分别以油红0染色、茜素红染色进行鉴定。
2.分别输注小鼠胎鼠、胎盘组织细胞悬液以及小鼠胎鼠、胎盘源间充质干细胞
我们首先腹腔注射6日龄BALB/c小鼠胎鼠组织细胞悬液以及孕18天BALB/c小鼠胎盘组织细胞悬液于不同组15月龄自然衰老雌性BALB/c老年小鼠腹腔。
其次分别尾静脉注射6日龄BALB/c小鼠胎鼠以及孕18天BALB/c小鼠胎盘源间充质干细胞于其它不同组15月龄自然衰老雌性BALB/c老年小鼠体内。
3.输注前后相关指标检测
输注后计数小鼠存活天数。输注前以及输注3个月后,超声心动图检查小鼠心输出量、每搏量、左心室舒张末容积、左心室质量；第二次超声检查完后首先血清学检测总超氧化物歧化酶活力、丙二醛含量以及谷胱甘肽过氧化物酶活力等抗氧化能力指标；其次检测反映心功能的心脏质量指数(心脏质量／体质量)、反映免疫功能的脾脏质量指数(脾脏质量／体质量)、反映消化道动力的结肠肌层厚度,解剖学检查干细胞致瘤性；并对心肌、肾脏、肺脏、皮肤等脏器做组织学衰老程度评分,最后对自然死亡小鼠Y染色体原位杂交检测植入的细胞。
结果：
1.小鼠胎鼠、胎盘源间充质干细胞符合间充质干细胞表面标志特点,成脂诱导后,油红0染色为阳性。成骨诱导后,茜素红染色也均为阳性。
2.小鼠胎鼠、胎盘组织细胞悬液输注组以及小鼠胎鼠源间充质干细胞输注组输注后的存活日期至少1.3倍于相应对照组,差异有统计学意义(均P<0.05)。
3.输注后未发现输注对小鼠造成不良影响,也未发现干细胞致瘤性；Y染色体原位杂交实验在所有输注组小鼠脏器检测到输注细胞长期存活。
4.小鼠胎鼠组织细胞悬液输注组、小鼠胎盘组织细胞悬液输注组以及小鼠胎鼠源间充质干细胞输注组分别与对照组心输出量减少量、每搏量下降量、左心室质量增加量、左心室舒张末期容积减少量、心脏质量指数、脾脏质量指数、结肠肌层厚度,心脏、肾脏、肺脏、皮肤等器官组织学衰老程度分值,血清总超氧化物歧化酶活力、丙二醛含量以及谷胱甘肽过氧化物酶活力,各指标的差异皆有统计学意义(均P＜0.05)。
5.小鼠胎盘源间充质干细胞输注组与对照组除了血清总超氧化物歧化酶活力、丙二醛含量以及谷胱甘肽过氧化物酶活力之间的差异有统计学意义外(均P<0.05),输注后存活日期等以上其它各指标的差异皆无统计学意义(均P＞0.05)。
结论：
1.小鼠胎盘中能分离出间充质干细胞。
2.小鼠胎鼠组织细胞悬液、小鼠胎盘组织细胞悬液以及小鼠胎鼠源间充质干细胞有抗小鼠衰老作用。
3.小鼠胎盘间充质干细胞没有明确的抗小鼠衰老功效。
Objectives:
To explore the anti-aging effect of the cells suspension and mesenchymal stem cells derived from murine fetus and placenta.
Methods:
1. Preparation of cells suspension of mouse fetus and placenta as well as isolation and identification of mesenchymal stem cells derived from them.
We got 6-day-old Balb/c mouse fetuses and 18-day-old Balb/c mouse placentas (female and male) with uterine-incision delivery. The tissue cells suspension of mouse fetus and placenta were prepared with repeated blow and inhalation, grind and filtration. The mesenchymal stem cells of mouse fetus and mouse placenta were isolated with density gradient centrifugation, two steps centrifugalization and adhesive screening methods. The cells were identified with cell-surface markers and cultured in an adipogenic medium or in an osteogenic medium. After induction, the cells were identified with oil red O staining and alizarin bordeaux staining respectively.
2. Implantation of cells suspension and mesenchymal stem cells.
Firstly,6-day-old fetus cells suspension and 18-day-old placenta tissue cells suspension were administrated respectively into abdominal cavity of 15-month-old naturally aging female BALB/c mice of different groups with intraperitoneal injection respectively.
Secondly, the mesenchymal stem cells from them were implanted respectively into 15-month-old naturally aging female BALB/c mice of other different groups with tail vein injection in other time.
3. Viewing objects before and after anti-aging treatment
The mice survival time were recorded after anti-aging treatment. The mice health situation were evaluated with ultrasoundcardiogram on cardiac output, stroke volume, left ventricle end-diastolic volume and left ventricular mass before and 3 month later after anti-aging treatment. Firstly, the serum total superoxide dismutase activity, serum maleic dialdehyde content and serum glutathione peroxidase activity were detected after the second ultrasoundcardiogram. Secondly the mice were evaluated with heart mass index representing heart function (heart mass/body mass), spleen mass index representing immune function (spleen mass/body mass), and colon muscular layer thickness representing digestive tract dynamia, and we detected the oncogenicity of stem cells with anatomy and evaluated the mice with score of cardia, kidney, lung, skin histopathology at the same time. At last the imbedded cells were traced in organ tissues of died mice with in situ Y chromosomal hybridization staining.
Results:
1. The cell-surface marker of mouse fetus and placenta derived mesenchymal stem cells was in accordance with that of mesenchymal stem cells. After induction the cells were positive for oil red O staining and alizarin bordeaux staining.
2. After implantation, the surviving time of mouse fetus tissue cells suspension group, mouse placenta tissue cells suspension group and mouse fetus derived mesenchymal stem cells group were at least 1.3 times of the corresponding control one, and the difference were significant (all P<0.05)
3. After implantation, we did not find any harmful effect with the implantation, nor any macroscopic tumor with all of postmortal anatomy inspection, but found the long-term surviving implanted cells located in organ tissues of all implanted mice with in situ Y chromosomal hybridization staining.
4. After implantation, many indexes showed the significant differences between mouse fetus tissue cells suspension implantation group, mouse placenta tissue cells suspension implantation group, mouse fetus derived mesenchymal stem cells implantation group and their corresponding control group, such as decrease of cardiac output, decrease of stroke volume, increase of left ventricular mass, decrease of left ventricle end-diastolic volume, heart mass index, spleen mass index, colon muscular layer thickness, the score of heart, kiney, lung, skin histopathology, serum total superoxide dismutase activity, maleic dialdehyde content and glutathione peroxidase activity (all P<0.05).
5. But there was no statistical significance on the difference of pre-stated indexes except for serum total superoxide dismutase activity, maleic dialdehyde content and glutathione peroxidase activity between mouse placenta derived mesenchymal stem cells implantation group and the control group.
Conclusions:
1. Mesenchymal stem cells can be isolated from mouse placenta.
2. Both mouse fetus derived cell suspension and mesenchymal stem cells and mouse placenta derived mesenchymal stem cells all have anti-aging capacity in mouse.
3. Mouse placenta derived mesenchymal stem cells has no conclusive anti-aging effect on mouse.
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