【作者】 王敏；

【导师】 孙新华；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2010， 博士

【摘要】 本实验的目的是识别miRNAs是人脂肪源性干细胞(human Adipose–Derived Stem Cells,hADSCs)成骨分化的一个调控因子,探讨对成骨分化有调节作用的寡核苷酸(oligodeoxynucleotide,ODN)是否通过调控miRNAs的表达从而调节成骨分化。从人脂肪抽吸物中分离hADSCs,体外培养扩增及鉴定。对hADSCs成骨分化的效率进行评估。检测miRNA-196a、microRNA-133a在hADSCs成骨分化中的表达以及Runx2、HOXC8的蛋白表达。将ODN C2及C12加入hADSCs成骨诱导培养基中诱导hADSCs成骨分化,以成骨诱导培养基诱导的hADSCs作为对照。检测三组成骨能力的变化,miRNA-196a、microRNA-133a表达的变化,Runx2、HOXC8蛋白表达的变化。结果证明hADSCs有极强的向成骨细胞分化的能力。miRNA-196a的表达在hADSCs成骨分化的过程中增强,伴随着HOXC8蛋白表达降低。提示miRNA-196a在hADSCs成骨分化中有重要作用,它的作用是通过HOXC8这一靶点介导的。通过本实验,miRNA-133a对成骨分化是否有调控作用尚不十分明确。本实验结果显示ODN C2及C12对成骨有抑制作用,是通过下调miRNA-196a表达水平,增加HOXC8的蛋白表达来实现的。本实验研究证明miRNAs是hADSCs成骨分化的调控因子;首次将ODN应用于hADSCs的成骨分化过程中,显示其对hADSCs的成骨分化有抑制作用;并证明ODN C2及C12对成骨的抑制作用是通过下调miRNA-196a表达水平,增加HOXC8的蛋白表达实现的。为hADSCs应用于临床作为组织工程骨的种子细胞及探索对成骨分化有影响的药物开发提供新的思路。更多还原

【Abstract】 Human Adipose–Derived Stem Cells (hADSCs) are excellent candidates for cell-based therapeutic strategies to regenerate injured tissue. Although hADSCs can be isolated from Adipose Tissue and directed to differentiate by means of an osteogenic pathway, the regulation of cell-fate determination is not well understood. MicroRNAs (miRNAs) are a class of small (～22 nt), noncoding RNAs that regulate gene expression at a post-transcriptional level. Recently, it has been shown that miRNAs have an influence on the complexity of the stemness state either by inhibiting the translation or by stimulating the degradation of target mRNAs. Luzi et al. reported that miR-26a inhibited osteogenic differentiation in hADSCs by targeting SMAD1. Oligodeoxynucleotides(ODNs)are a class of small (less than one hundred nucleotides), single strand DNAs that mimic bacterial or human DNA. DNA is a kind of biomacromolecule which is composed of nucleotides; almost all the genetic information determining biological features is stored in DNA molecule. The sequence character and modified style of DNA varies significantly between different species. Recent studies demonstrate that DNA has some special biological activities in addition to carrying and transmitting genetic information, for example, certain DNA with specific sequence and modification has stimulatory or inhibitory effect on the immune system. Some DNA which has specific sequence and modification can be used for the prevention and treatment of diseases. CpG ODN, upon TLR9 ligation, modulates osteoclastogenesis via interactions with osteoclast precursors or induction of osteoblasts osteoclastogenic activity. Some CpG ODNs affect the proliferation of hADSCs and decrease the osteogenic differentiation. Recently, the members of our research group have shown that ODNs with special sequence characteristic can significantly promote the differentiation of rat BMSCs to osteoblasts. The aim of this study is to determine whether microRNAs serve as regulators of the osteogenic differentiation of hADSCs and whether the osteogenic differentiation of hADSCs is affected by ODNs through its regulating of microRNAs expression. This study includes four aspects:1. Cell cultures and osteogenic differentiation of hADSCshADSCs were isolated from adipose tissue obtained from the subcutaneous abdominal depot undergoing elective abdominoplasty in accordance with a protocol approved by the Institutional Review Board for human studies. The isolation procedure was according to the methods described in the previous studies of Zuk’. All the cells used in the experiments were in three passages. The hADSCs were cultured and amplified in vitro and were identified by cell phenotype detection. Multi-lineage differentiation potential was assessed by histological staining such as oil red O for lipid accumulation and alizarin red S for mineralization nodules during adipogenic and osteogenic induction. Osteogenic differentiation induction group was divided into five subgroups: 0, 7, 14, 21 and 28 d groups. Alizarin red S staining, Van Gieson collagen fiber staining and ColⅠimmunofluorescence were performed to detect the expression of ColⅠ. FV Viewer 1.7 software was employed to analyze the date. The positive stainning of oil red O was found 14 d after adipogenic induction. For the osteogenic induction groups, the calcified nodules and the collagen fiber red staining was found in 21 d group. The expression of ColⅠwas found in 21 d group for most cells and was found strongly positive expression in 28 d group. Compared with 0 d group, the expression of ColⅠin osteogenic induction group was significantly increased from 7 d to 28 d(P<0.05). This aspect of the study has shown that osteogenic induction of hADSCs, most of the cells have osteogenic capability and the hADSCs have outstanding capability of osteoinductive and osteogenesis. hADSCs may be useful in future clinical cell-based therapy for bone regeneration engineering.2. The expression of microRNAs and osteoblastic marker genes of hADSCs during osteogenic differentiationOsteogenic differentiation of hADSCs was induced for 28 days. MiRNAs were prepared at the indicated times and relative real-time PCR were performed to detect the miRNAs. U6 was used as a loading control and for normalization. The relative quantities of the amplified products were determined with an image analyzer. The data represent the mean±SE of the relative ratio of the miRNAs signal of the corresponding samples (n = 3). The expression of Runx2 and ColⅠmRNA was evaluated after the induction by simultaneous monitoring the expression of genes involved in osteoblastic differentiation by relative real-time RT-PCR, normalized toβ-actin. Relative levels are expressed setting 0 time to 1. MiR-196a expression was increased and miR-133a expression up-regulated to 7 d, then decreased during osteogenic differentiation of hADSCs. Runx2 mRNA expression was increased and had a significant increase at 7 d during hADSCs differentiation. ColⅠmRNA had two peak at 3 d and 14 d, and had a significant decrease at 7 d during hADSCs differentiation. Our data indicate that miR-196a plays an important role in hADSCs osteogenic differentiation and miR-133a may also play a role in hADSCs osteogenic differentiation. The osteoblasts differentiated from hADSCs have osteoblastic potential to express Runx2 and ColⅠmRNA.3. The protein expression of targets of microRNAs and osteoblastic markers of hADSCs during osteogenic differentiationTotal protein extracts were prepared at the indicated times. Runx2 and HOXC8 proteins expression were evaluated by Western blotting analysis. GAPDH was used as the standard protein. The protein expression of Runx2, served as markers for osteogenic differentiation, was observed at all time-points in osteo-induced hADSCs. Furthermore, Runx2 basal expression was observed in noninduced hADSCs. Runx2 proteins expression was increased and had a significant increase at 7 d during hADSCs differentiation. HOXC8 protein expression was down-regulated during hADSCs osteogenic differentiation, whereas expression of miR-196a was complementary to that of HOXC8. Our data indicate that miR-196a plays an important role in hADSCs osteogenic differentiation and proliferation, which may be mediated through its predicted target, HOXC8.4. The effect of ODN C2 and ODN C12 on microRNAs modulation of hADSCs osteogenic differentiationTo investigate the effect of ODN addition during osteogenic differentiation induction, ODN C2, ODN C12 was added to the osteogenic medium and hADSCs osteogenic induction were performed in three groups: ODN C2 added to osteogenic medium( I+C2) group, ODN C12 added to osteogenic medium( I+C12) group, osteogenic medium(I )group. The effect was evaluated by means of histological observation, genomic and protein measurement. Alizarin red S staining and ColⅠimmunofluorescence were used as well as direct observation. The expression of Runx2 and ColⅠmRNA were evaluated by relative real-time RT-PCR. ALP proteins expression was evaluated by ALP Substrate measurement and Runx2 proteins expression was evaluated by Western blotting analysis. MiRNAs were prepared at the indicated times of the three groups and relative real-time PCR were performed to detect miR-196a and miR-133a expression. HOXC8 protein expressions were evaluated by Western blotting analysis. The calcified nodules and the ColⅠimmunofluorescence of I+C2, I+C12 group were less than I group. The Runx2 and ColⅠmRNA of I+C2, I+C12 group were less than I group. The miR-196a and miR-133a expression of I+C2, I+C12 group were less than I group in 3, 7, 14, 21 d. The HOXC8 protein expression of I+C2, I+C12 group were more than I group. ODN C2, ODN C12 promoted the proliferation of hADSCs and decreased the osteogenic differentiation. ODN C2, ODN C12 decreased the expression of miR-196a and miR-133a during hADSCs differentiation. ODN C2, ODN C12 down-regulate the osteogenic differentiation of hADSCs by repressing the expression of miR-196a and thereby releasing the target HOXC8 protein expression.Taken together, our results suggest that hADSCs cultured in vitro have outstanding capability of osteoinductive and osteogenesis. HADSCs may be useful in future clinical cell-based therapy for bone regeneration engineering. Our data indicate that miR-196a plays a role in hADSC osteogenic differentiation, which may be mediated through its predicted target, HOXC8. MiR-133a plays a role in hADSCs differentiation. ODN C2, ODN C12 promoted the proliferation of hADSCs and decreased the osteogenic differentiation. ODN C2, ODN C12 down-regulate the osteogenic differentiation of hADSCs by repressing the expression of miR-196a and thereby releasing the target HOXC8 protein expression.The innovation of this research is that the expression of miR-196a and HOXC8 has been monitored consecutively during 28 days, a complete period of hADSCs osteogenic differentiation. The first time that ODN C2, ODN C12 has been added to the osteogenic medium to investigate the influence on hADSCs osteogenic differentiation and miRNAs expression. Our results suggest that miRNAs functions as a regulator of osteogenic differentiation by repressing target gene expression. It is suggested that some drugs, like ODN with special sequence, inhibited osteogenic differentiation by regulating miRNAs expression. Elucidation of the molecular mechanisms guiding human adipose–derived stem cells (hADSCs) differentiation and obtaining drugs regulated this process are of extreme importance for improving the treatment of bone-related diseases such as alveolar bone defects, maxillofacial defects, periodontitis and osteoporosis.更多还原