软骨内成骨过程中PTHrP旁分泌调节HDAC4细胞核内外穿梭调控生长板内软骨细胞增殖的机制研究

The longitudinal growth of the limb comes from the growth plate, where the initial step of chondrocyte proliferation is critical since it sets the overall maturation process for endochondral bone formation, is a determinant of size and shape of long bone and, when abnormal, can lead to skeletal defects. Despite its obvious importance, relatively little is known about its regulation (e.g., what triggers the onset, and end of chondrocyte proliferation? which factors regulate it and how do they act?). Recent studies from our and other laboratories have now shown that HDAC4 may play a critical role in growth plate development and have a specific distribution pattern, which is associated with the chodrocyte proliferation during skeletogenesis in growth plate in our lab, but it is not clear what regulates the specific distribution pattern of HDAC4 itself and how the distribution pattern of HDAC4 affects chondrocyte proliferation..Studies from the applicant, and other laboratories have now shown that entry of HDAC4 into the nuclei is associated with chondrocyte proliferation; PTHrP may drive nuclear entry of HDAC4 through the Protein Kinase A pathway to regulate chondrocyte proliferation and differentiation in the growth plate, which induces cell proliferation and inhibits chondrocyte hypertrophy by increasing PCNA and suppressing Runx2 in proliferating chondrocytes. Therefore, we assume that PTHrP promote HDAC4 into the nucleus to promote chondrocyte proliferation by paracrine regulatory mechanisms. To confirm this hypothesis, we design following experiment in the animal models: using the PTHrP knockout mice and COL-Ⅱ-PTHrP transgenic mice to explore the mechanism of PTHrP promoting HDAC4 into the nucleus; interventing cultured mouse chondrocytes by GFP-HDAC4 and Flag-PCNA, combining with HDAC4 knockout mice experiments, explore the mechanism on HDAC4 promote chondrocyte proliferation. In conclusion. It is suggested that these results will provide much needed information on what regulates the specific distribution pattern of HDAC4 itself and how the distribution pattern of HDAC4 affects chondrocyte proliferation.

软骨细胞增殖障碍是导致关节发育畸形和矮小畸形的重要原因，但由于软骨细胞增殖机制不清，因此临床尚缺乏有效的治疗方法。我们前期研究发现，生长板内软骨细胞核中HDAC4蛋白分布规律与甲状旁腺激素相关多肽（PTHrP）蛋白的分布规律一致，均与软骨细胞增殖能力呈正相关，且PTHrP可使HDAC4进入细胞核与增殖细胞核抗原（PCNA）结合，进而促进软骨细胞增殖，但具体机制不清。因此假设：PTHrP通过旁分泌调节机制促进HDAC4进入细胞核与PCNA相互作用促进软骨细胞增殖。为此，本研究拟利用PTHrP基因敲除小鼠及PTHrP基因过表达小鼠，探讨PTHrP促进HDAC4进入细胞核的机制；利用HDAC4、PCNA及羟基脲干预离体培养的小鼠软骨细胞，结合HDAC4基因敲除小鼠及HDAC4过表达小鼠的在体实验，探讨HDAC4促进软骨细胞增殖的机制。该假说的证实将为软骨发育畸形及矮小畸形患者的治疗提供新方向。

软骨细胞增殖障碍是导致关节发育畸形和矮小畸形的重要原因，但目前软骨细胞增殖机制不清，尚缺乏有效治疗方法。前期发现，生长板内软骨细胞核中去乙酰化酶4（HDAC4）与甲状旁腺激素相关多肽（PTHrP）分布规律一致，均与软骨细胞增殖能力呈正相关，因此假设：PTHrP促进HDAC4进入细胞核与PCNA相互作用促进软骨细胞增殖。本研究：1. 利用外源性PTHrP（过表达PTHrP）、PTHLH-homo-617质粒（抑制PTHrP表达）转染离体培养的软骨细胞，并对软骨细胞中HDAC4亚细胞定位进行检测；结果显示过表达PTHrP基因可促进HDAC4由胞浆穿梭入胞核；2. 利用HDAC4腺病毒转染软骨细胞发现细胞核中HDAC4可促进软骨细胞增殖，利用不同HDAC4片段质粒（1-289，1-326，1-699，629-1040及full-HDAC4 干预软骨细胞，结果显示HDAC4促进软骨细胞增殖的机制与HDAC4和PCNA之间的结合密切相关，HDAC4 蛋白326-669之间可能存在一个与PCNA结合的位点。3. 利用HDAC4基因敲除小鼠制备模型，结果显示HDAC4表达量降低可降低软骨细胞增殖，促进软骨细胞肥大。4. 关节腔注射HDAC4腺病毒，在体过表达HDAC4可有效延缓大鼠软骨损伤程度促进软骨细胞合成代谢。本研究结合体内及体外实验对假说进行了证实。截止目前为止，本项目已资助发表SCI文章8篇，中文文章4篇，申请国家专利2项，选派3名课题组成员赴美国交流学习，参加国际会议20余人次。