| Cells transplantation was regarded as a rather effective treatment means for diseases presently, such as Parkinson's Disease (PD), Alzheimer's Disease (AD), caused by some specific cells injuries or absence. Along with the technique breakthrough of somatic cell nuclear transfer and stem cells, therapeutic cloning (TC) was employed in the cells transplantation treatment. It is possible that totipotent embryonic stem cells (ESCs) with self-genetic information will be generated by neuclear transfer of normal somatic cells of individual. Development and perfection of therapeutic cloning can offer a new approach for seeking new seed cells used for biological tissue engineering research. The ESCs derived from TC can resolve the problems of both cellular source limitation and immunorejection.Object: To get and examine the developmental ability in vitro of therapeutic cloning embryos established by neuclear transfer using human bone marrow stromal cells (hBMSCs).Methods and Results: Some serial researches were performed in the experiment, including filtrating the superovulation ways in rabbits so as to obtain numerous and efficient oocytes, isolating and culturing hBMSCs, studying their morphological characterization related to the cloning, and investigating the appropriate activation ways and culture system in vitro of rabbit parthenogenetic embryos so as to make an experimental foundation for TC. Finally, the reconstructed embryos were obtained between hBMSCs and enucleated rabbit oocytes. The main contents and results were shown as following.1,Superovalation of rabbit ooeytesCompare the superovalation effect between pregnant mares serum gonadotropin (PMSG) and Follicle-Stimulating Hormone (FSH) in New Zealand Rabbits. At 72h after injection 50IU PMSG in muscle, 150IU Human Chofionic Gonadotropin (HCG) were intravenously injected in PMSG group, Following six of consecutive muscle injections of 0.15rag FSH/rabbit/time given 12 hours of apart. time for 3 days, 150IU of HCG were intravenously injected after the final dose of FSH. At the 14～15th hour after HCG injection, the mature oocytes were flushed out from the oviducts with M199 (TCM-199+10% FBS) or aspirated from the follicles of the ovaries.Compare the superovalation effect between FSH constant injection group and FSH descending injection group. With six consecutive muscle injections of 0.15mg FSH/rabbit/time given 12 hours of apart time for 3 days in constant injection group. With six consecutive muscle injections of FSH given 12 hours of apart time for 3 days in descending injection group, the first and second doses were 0.15mg FSH/rabbit, the third and fourth doses were 0.1mg FSH/rabbit, while the fifth and sixth doses were 0.05mg FSH/rabbit. 150IU of HCG were intravenously injected at the 12th hour after the final dose of FSH in both of two groups.Result:(1) Mature oocytes obtained from ovduction flushing (ovulated) and follicle aspiration (aspirated) respectively were 10.42±4.3, 12.47±5.6 in PMSG group, and 20.8±10.1, 14.64±13.0 in FSH group. The results showed that ovulated oocytes have significant difference between PMSG and FSH groups (T=-4.663, P=0.000).(2) The numbers of oocytes by the ovulated or aspirated, following the FSH descending treatment, were respectively 20.94±8.3 and 13.73±14.3. Whereas the average number of oocytes obtained by the ovulated or aspirated, following the constant treatment of FSH, were respectively as 20.8±10.1 and 14.64±13.0. No difference in the counterparts of oocytes between ovulating and aspirating no matter in contant or descending groups of FSH treatment.2. Culture and biological characteristics of hBMSC related the cloninghBMSCs were isolated by density gradient centrifugation with percoll and cultured in vitro at 37℃, 5%CO3. By detected, the hBMSCs showed the similar appearances, such as the long spindle shape, and the circular or oval nucleus. The average diameter of hBMSCs was about 25～28μm following the digestion of 0.25% trypsin. The cells showed a stable growth and high percentage (about 92.5%) in G0/G1 phage. The cultured hBMSCs possessed normal morphology and cytoskeleton with immunofluorescence detection, and their telomerase activity was 0.567. Karyotype examination of the hBMSCs by Giemsa staining at the 7th passage showed that more than 90% cells had normal human chromosome numbers (2n=46), without abnormal chromosome.3,Parthenogenetic activation of rabbit oocytes and culture of reconstructed embryos(1) The survival rate of oocytes actived in 51μmol/L ionomycin (ION) for 4 min were significantly higher than that of ones stimulated by 2.0 kv/cm electricity treatment (p=0.016). However no significant difference in the survived rate of oocytes between the electric activity with 1.2kv/cm and 1.6 kv/cm treatment and the ION treatment. The percentages of the cleaved oocytes, developing 8-16 cells, and the blastocysts were also significantly higher in the ION treated group, comparied with the electric treated group by 1.2kv/cm treatment (p=0.000, p=0.002, p=0.026).(2) To investigate the effect of various concentration of leukemia inhibitory factor (LIF) or Insulin+Transferrin+Sodium selenite (ITS) on development of parthenogenetic embryos cultured in M199.The activated oocytes were cultured in M199 supplemented with various concentrations (0, 10, 20, 40 and 80 ng/ml ) of LIF. The results showed no any effect of LIF on the rates of both cellular cleavage and 8-16-cell forming. Whereas, the rate of blastocysts showed a significant difference (p=0.003) between 20 ng/ml and 80 ng/ml of LIF supplementation. In addition, total number of blastocysts in the medium supplemented with 20ng/ml LIF was significantly different compared with 40ng/ml and 80ng/ml of LIF respectively (P=0.011, P=0.002), but no significant difference compared with 0 ng/ml LIF and 10 ng/ml LIF.The activated oocytes were cultured in M199 supplemented with various concentrations (0×, 1×and 2×) of ITS. The results showed no effect of ITS on the rates of both cellular cleavage and the 8-16-cell forming. However, the rate of blastocysts showed a significant difference (p=0.003) between 1×and 2×supplementation of ITS. In addition, total cell number of blastocysts in the mediumsupplemented with 0×or 1×was significantly different compared with 2×ITS (P=0.015, P=0.044).4. Establishment of reconstructed embryos by nuclear transferTo obtain the reconstructed embryos by nuclear transfer, the hBMSCs were cultured as the nuclear donors and enucleated rabbit oocytes as the nuclear recipents. 277 of mature oocytes were enucleated and then injected new nuclei from hBMSCs. The success percent of nuclear transfer was 66.43 % (184/277) and fusion rate was 53.8% (99/184). After cultured in vitro, the rates of the cellular cleavage and blastocyst forming were 87.88% (87/99) and 4.6% (4/87) respectively. 100% (4/4) of blastocysts obtained from cloning were developed to hatching stage.ConclusionTherapeutic cloning embryos can be produced via nuclear transfer by using hBMSCs as nuclear donors and rabbit enucleated oocytes as the nuclear recipents. The results indicate that the enucleated oocytes used in this study can support hBMSC's nuclei to re-program, furthermore, the cloned embryos possess the potential developmental ability to the further stage. |
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