| With the development of human society, the incidence of some diseases in human beings resulted from central nerve systemic (CNS) deterioration such as Parkinson’s disease (PD), Alzheimer’s disease (AD) is on rise, and the rate of CNS trauma such as traumatic brain injury (TBI), spinal cord injury (SCI) are also on rise. To date, all the methods include pharmacy and surgery therapy can not cure these diseases essentially, therefore, it is necessary to explore new ways to cure them. The advent of regenerative medicine of stem cells, especially the substitute therapy by cellular, tissue and organs’transplantation, provides a new prospection for the therapy of CNS deteriorative and traumatic diseases. With the technological breakthrough of mammal somatic cellular nuclear transfer (SCNT) and human embryonic stem cells (hESCs) research, the concept of therapeutic clone has been proposed. The patient-specific ESCs harvested by SCNT, can be induced and differentiated into various cells to substitute or repair the degenerated cells with age or traumatic, even to cure some diseases. The low efficiency of SCNT, however, blocked the development of TC. Therefore, to explore how to improve the rate of blastocst of cloned embryos reconstructed by SCNT is a critical matter and would be a hotspot in the domain of stem cells therapy and application. It includes the type and differentiated stage of donor cells, the chemical treatment by cloned embryos, and the methods improvement of nuclear transfer etc.Object To improve the efficiency of SCNT. In this research, we investigated the effect of different diameter enucleation/injection pipettes and two different methods on the early development of embryos reconstructed by SCNT using a pizeo-activated micromanipulator. Otherwise, we also studied the effect of various concentration of scriptaid on the development of cloned embryos.Methods Firstly, we compared the effect of different diameter enucleation/injection pipettes (5～6μm、7～8μm、9～10μm) on cloned embryos reconstructed by SCNT, and selected the best pipettes to SCNT. Secondly, we carried out SCNT using two different methods, named "one-step" and "two-step" respectively according to the different procedure of enucleation and injection, and studied the effect of two methods on the development of cloned embryos. Finally, we treated the cloned embryos with various concentration of scriptaid and observed the effect of scriptaid on the early development of cloned embryos reconstructed by SCNT.1. Using three different inner diameters of pipettes for enucleation and injectionAfter being collected, good oocytes were divided randomly into three groups and enucleated using different inner diameter pipettes (5～6μm、7～8μm、9～10μm). Then, we recorded and compared the survival rate of enucleated oocytes. According to the first result, the enucleation of oocytes was performed by the best pipette. All the enucleated oocytes were divided into three groups and injected by using different inner diameters of pipettes (5～6μm、7～8μm、9～10μm) with cumulus as donors. We then recorded and compared the survival rate of cloned embryos.2. Reconstructing the cloned embryos by SCNT with two methodsAfter being collected, all good oocytes were divided into two groups. Group One, named "two-steps" procedure, in that the spindles were removed firstly and the enucleated oocytes were recovered for30min at least, and then the cumulus cells were injected using micromanipulator system. Group two, named "one-step" procedure, in that the oocytes enucleation and the donor-nuclear injection were synchronously performed. We counted and compared the time and effectiveness of SCNT by these two different procedures, recorded the survival of cloned embryos respectively. All the cloned embryos were activated by10mM SrCl2and then cultured in incubator at the classifying of different groups respectively. We then observed and recorded the development of cloned embryos, also compared the rates of activation,2-cell cleavage, and blastocysts of cloned embryos to assess the effect of two methods on the development of cloned embryos in vitro.3.Treating the cloned embryos by various concentration of ScriptaidAll the cloned embryos, reconstructed using two-steps methods of SCNT, were divided randomly into five groups and activated respectively in Ca2+-free-CZB activation medium with respective OnM,50nM,100nM,250nM, and500nM Scriptaid for6h. And then, they were treated again correspondly in KSOM medium with OnM,50nM,100nM,250nM, and500nM Scriptaid for another4h. After the treatment, all the cloned embryos were cultured in KSOM medium according to groups for96h. We observed and recorded the rates of activation, cleavage of2-cell, blastocysts during the development of cloned embryos in vitro, and compared the effect of Scriptaid on the early development of cloned embryos.Statistical analysisAll the sample data were analyzed statistically by SPSS13.0software. In part1, the data of three groups were analyzed by Chi-square test with the total test level a=0.05and correction test level a=0.0125. In part2, the data of two groups were analyzed by Chi-square test with the test level a=0.05, and the mean time of two methods was represented by "mean±SD" and was analyzed by indepent-sample t test with the test level a=0.05. In part3, the data of five groups were analyzed by Chi-square test with the total test level a=0.05and correction test level a=0.00455. The number cells of blastocysts were represented by "mean±SD", and were analyzed totally by one-way ANOVA with test level a=0.05, and were multi-compared between any two groups by LSD-t.Results1. Comparison the survival rates of enucleated oocytes/cloned embryos following the SCNT by different inner diameter pipettes1.1In the enucleation research,124,130and115of good oocytes were randomly selected into the three groups with the pipette inner diameter as5-6μm,7～8μm,9～10μm respectively. After enucleation, the survival rates of oocytes were as64.5%(80/124),92.3%(120/130) and89.6%(103/115) respectively. There was a significant difference among the survival rate of oocytes enucleated by different inner diameter enucleation pipettes (χ2=39.691, P<0.001). There is a significant difference between5～6μm group and other two groups (χ2=29.282, P<0.001and x2=20.867, P<0.001, respectively). However, there was no significant difference between Group7～8μm and Group9～10μm(χ2=0.562, P=0.454). These results suggested that the pipettes with inner diameter7～8μm and9～10μm could get the better survival rate of enucleated oocytes than that with inner diameter5～6μm did1.2In the injection research, three groups (pipettes inner diameter as respective5～6μm,7～8μm, and9～10μm) randomly possessed107,110and115of health enucleated oocytes. After being injected cumulus cells, the recombination cloned embryos showed the survival numbers as89,96and71respectively, and the survival rates of cloned embryos were respectively as83.2%(89/107),87.3%(96/110) and61.7%(71/115). The survival rate of cloned embryos among three groups showed a significant difference (χ2=24.061, P<0.001). Group9～10μm exhibited a significant difference from other groups (x2=12.656, P<0.001; x2=19.158, P<0.001). However, there was no significant difference between Groups5～6μm and7～8μm (χ2=0.724, P=0.395). These suggested that the pipettes with inner diameter5～6μm and7～8μm resulted in a better survival rate of cloned embryos than that with9～10μm did.2. Comparison of two methods of nuclear transferThe surivival rate of cloned embryos reconstructed by "two-steps" and "one-step" were77.2%(61/79) and85.1%(63/74) respectively, there was no significant difference between the two groups (χ2=1.560, P=0.212). The mean time of reconstructing cloned embryos by "two-steps" and "one-step" were131.57±10.88s and74.53±11.77s respectively. There was a dramatic significant difference between the two groups (t=31.148, P=0.000). It suggested that "one-step" could reconstruct the cloned embryos with only the fewer time and the faster speed than "two-steps" did with. The activation rate and2-cell cleavage rate of cloned embryos reconstructed by "two-steps" and "one-step" were65.6%VS85.7%and55.0%VS75.9%respectively, there were dramatic significant difference between the two groups (χ2=6.855, P=0.009and χ2=4.552, P=0.033respectively). It suggested that "one-step" was the better than "two-steps" on the activation rate and2-cell cleavage rate of cloned embryos. However, the blastocyst rate of cloned embryos cultured in vitro by two methods were as9.1%and12.2%respectively. There was no significant difference between the two groups (χ2=0.145, P=0.704), It implied that the two methods had little influence on the blastocyst of cloned embryos in vitro.3. Effect of various concentration of Scriptaid on the early development of cloned embryos in vitroAll the cloned embryos, upon the concentrations of Scriptaid, were divided randomly into five groups (OnM,50nM,100nM,250nM,500nM) and activated in Ca2+-free-CZB activation medium with various concentration of Scriptaid for6h. Then the cloned embryos were transferred into KSOM medium with various concentration of Scriptaid respectively, and then incubate for another4h. After being treated, they were cultured in KSOM medium respectively for96h. The rate of activation and2-cell cleavage of cloned embryos showed no dramatic significant difference in these groups (x2=0.574, P=0.966and χ2=1.344, P=0.854). The blastocyst rate of cloned embryos showed a significant difference in the five groups (x2=15.921, P=0.003). However, there was no significant difference among other groups. The best blastocyte rate was the group with250nM Scriptaid (24.2%), which showed approximately4.6times than that with OnM group (control group without Scriptaid,5.3%). The number of blastocyst also had a significant difference in five groups. The number of blastocyst in Groups OnM and250nM showed the significant difference (44.67±1.53and56.27±2.43respectively). The number of blastocyst in Group250nM exhibited the dramatic significant difference t from others. It suggested s that the cloned embryos treated by250nM Scriptaid had much better blastula and quality of blastocyst than that treated in other groups, and250nM Scriptaid could improve the early development of cloned embryos in vitro.ConclusionThe inner diameter of enucleation/injection pipettes related to the successful clone embryos reconstructed by SCNT. It suggested that we should select better pipettes to enucleate and inject during the SCNT. The efficiency between the "two-steps" and "one-step" existed a significant difference on the cloned embryos reconstructed by SCNT, however, the blastocyte rate of cloned embryos showed no significant difference. Scriptaid, a histone deacetylase inhibitor, could enhance the blastocyst rate and blastocyst quality of inbred mice cloned embryos reconstructed by SCNT, and improve the early development of cloned embryos in vitro.250nM Scriptaid has a best effect on the early development of cloned embryos in vitro in inbred mice C57/BL6. |
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