王榮躍 魯文潔 邱海凡 戴芬
[摘要] 目的 研究CDK13基因?qū)θ毖跣阅X損傷小鼠細(xì)胞凋亡的影響。 方法 將野生型和CDK13基因敲除型小鼠分別分為野生型假手術(shù)組(SWT)、野生型模型組(MWT)、基因敲除假手術(shù)組(SKO)及基因敲除模型組(MKO)。采用PCR反應(yīng)法檢測CDK13基因表達(dá)、TTC染色法檢測腦梗死程度、免疫組化法檢測腦組織中活化型半胱天冬酶-3(CC3)表達(dá)、TUNEL法檢測細(xì)胞凋亡情況,四組小鼠進(jìn)行相關(guān)指標(biāo)比較。 結(jié)果 野生型模型組(MWT)小鼠腦組織梗死程度較基因敲除模型組(MKO)小鼠明顯減輕(P<0.05),且腦組織凋亡陽性細(xì)胞數(shù)明顯減少,凋亡指數(shù)降低,凋亡蛋白CC3表達(dá)減少(P<0.05)。 結(jié)論 敲除CDK13基因具有加劇小鼠缺氧性腦損傷的作用。
[關(guān)鍵詞] CDK13基因;基因敲除;細(xì)胞凋亡;模型
[中圖分類號] R722.1? ? ? ? ? [文獻(xiàn)標(biāo)識碼] A? ? ? ? ? [文章編號] 1673-9701(2019)34-0039-04
Effect of CDK13 gene on apoptosis in mice with hypoxic brain damage
WANG Rongyue1? ?LU Wenjie2? ?QIU Haifan1? ?DAI Fen1
1.Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China; 2.Outpatient Department, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou? ?325000, China
[Abstract] Objective To study the effect of CDK13 gene on apoptosis in mice with hypoxic brain injury. Methods Wild type and CDK13 knockout mice were divided into wild type sham operation group (SWT), wild type model group (MWT), gene knockout sham operation group (SKO) and gene knockout model group (MKO). The expression of CDK13 gene was detected by PCR reaction. The degree of cerebral infarction was detected by TTC staining. The expression of activated caspase-3 (CC3) in brain tissue was detected by immunohistochemistry. The apoptosis was detected by TUNEL method. Four groups of mice were compared. Results Compared with the MKO group, the degree of cerebral infarction in the MWT group was significantly reduced (P<0.05), and the number of apoptotic positive cells, apoptotic index and CC3 expression were significantly decreased(P<0.05). Conclusion Knockout of CDK13 gene aggravates hypoxic brain damage in mice.
[Key words] CDK13 gene; Gene knockout; Apoptosis; Model
智力障礙(mental retardation,MR)是指出現(xiàn)在18周歲之前以認(rèn)知障礙和社會適應(yīng)能力缺陷為主要表現(xiàn)的一種常見的出生缺陷疾病,在世界范圍內(nèi)患病率約為1%~3%[1,2]。智力障礙嚴(yán)重危害兒童及青少年的身心健康,MR具有終生發(fā)病和不可治愈的特點,目前研究認(rèn)為細(xì)胞壞死及凋亡是神經(jīng)元細(xì)胞死亡的主要方式,同時研究發(fā)現(xiàn)腦缺氧神經(jīng)元損傷有多種死亡機(jī)制參與[3,4],但細(xì)胞凋亡發(fā)生的具體機(jī)制仍不清楚。
CDK13(cyclin-dependent kinases 13,CDK13)是最晚被發(fā)現(xiàn)的細(xì)胞周期依賴性激酶家族成員,該家族蛋白C端有一段20個ATP依賴的絲氨酸-蘇氨酸蛋白激酶結(jié)構(gòu),通過整合細(xì)胞內(nèi)外的信號,實現(xiàn)細(xì)胞循環(huán)和基因轉(zhuǎn)錄過程的調(diào)控功能。CDK12和CDK13具有較多的同源序列,最近發(fā)現(xiàn)其可能在轉(zhuǎn)錄和加工RNA過程中發(fā)揮重要作用[3,4]。人類CDK13蛋白分子量較大,為165 kDa,已知CDK13與周期蛋白Cyclin K結(jié)合形成蛋白復(fù)合物可發(fā)揮生物學(xué)功能[6,7]。CDK13通過磷酸化絲氨酸蛋白酶Omi/HtrA2促進(jìn)細(xì)胞凋亡[8],在前期研究中發(fā)現(xiàn)CDK13基因缺失可導(dǎo)致智力障礙[9],推測CDK13可能參與腦缺氧過程引起細(xì)胞凋亡。因此本研究以缺氧性腦損傷作為研究區(qū)域,采用線栓法建立新生鼠(MCAO)模型,探討CDK13基因敲除對小鼠缺氧性腦損傷細(xì)胞凋亡的影響。
2017年,Bostwic BL等[18]對一組CDK13新發(fā)突變患兒的臨床表型進(jìn)行深入分析,發(fā)現(xiàn)CDK13變異可致患兒發(fā)生先天性心臟病、面容異常及智力發(fā)育障礙,但具體分子機(jī)制仍不詳。本研究發(fā)現(xiàn),兩組假手術(shù)組小鼠之間神經(jīng)元凋亡數(shù)目無差異,而CDK13基因敲除后凋亡明顯增加,提示CDK13基因敲除后可能通過激活其他路徑上的蛋白加重腦損傷,如JNK[20,21]、p53[22]、NF-κB[23,24]等,其在細(xì)胞凋亡中可能起重要作用,對深入闡明CDK13敲除引起小鼠腦組織損傷的作用機(jī)制具有重要意義。CDK13缺失可能導(dǎo)致小鼠突觸前谷氨酸等興奮性遞質(zhì)的過度釋放,激活p38MARK,通過p21等表達(dá)影響細(xì)胞凋亡,降低缺氧小鼠腦神經(jīng)細(xì)胞損傷。本研究同時觀察到敲除CDK13后能降低腦神經(jīng)細(xì)胞凋亡的發(fā)生,與Wu HJ等[25]的報道基本符合。本研究發(fā)現(xiàn)CDK13基因敲除后小鼠腦組織凋亡程度較重,且凋亡蛋白表達(dá)明顯增加,提示神經(jīng)細(xì)胞損傷增加,但這一路徑的中間過程仍有待下一步深入研究。
綜上所述,敲除CDK13基因具有加劇小鼠缺氧性腦損傷的作用,為明確智力障礙的原因提供了新思路,但具體分子機(jī)制需進(jìn)一步研究闡明。
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(收稿日期:2019-06-17)