腺相关及腺病毒内耳基因转染和Rb敲除结合ISL1表达诱导小鼠毛细胞与支持细胞增殖

发布时间：2018-05-14 02:03

本文选题：腺病毒 + 腺相关病毒　；参考：《复旦大学》2012年博士论文

【摘要】：低等脊椎动物如鸟类、鱼类的内耳毛细胞损伤后能够终生再生,而哺乳动物耳蜗毛细胞无自发性再生能力,因此毛细胞丢失所导致的感音神经性耳聋目前仍是临床治疗中的一大难题。另外一方面,上百种基因突变引起的遗传性耳聋目前没有治疗方法。因此,研究毛细胞再生以及内耳基因导入方法的发展成为重获听觉的焦点。分子生物学研究的发展为毛细胞再生带来了希望,内耳的基因治疗和外源性基因的导入,为耳聋的治疗带来了新的手段。腺病毒及腺相关病毒是内耳基因治疗常见的载体。为了验证合适的基因治疗的载体,我们使用新近发展的纳升级显微操作导入系统,大范围的在体研究了12种不同血清型的AAV作为基因载体导入的可行性,我们发现在新生鼠和成年鼠中,不同血清型的AAV可以转染不同的内耳感觉上皮细胞,包括支持细胞和毛细胞。我们还发现在新生鼠(P1/P2)给予内耳注射AAV可以持续表达较长时间,而对听力的影响很小(0-15dB)。这种方法可以用来作为早期内耳发育过程中基因功能缺失所致的遗传性耳聋的基因治疗手段。在胚胎期的内耳毛细胞及支持细胞的前体细胞中选择性敲除Rbl可产生数量远超出正常情况的前体细胞,这些细胞可进一步分化成毛细胞和支持细胞。但Rb1敲除后诱导毛细胞和支持细胞增殖是年龄相关性的,其具体引起增殖的时间截点如何?即Rbl敲除对不同年龄段内耳感觉上皮细胞重新进入细胞周期的功能如何?是否可以结合其他内耳前体细胞基因可以让更成熟甚至成年的哺乳动物毛细胞、支持细胞增殖?本研究拟行5型腺病毒经中阶路径注射转染新生和成年小鼠耳蜗毛细胞和支持细胞的评价；以Er-Cre Rb loxp/loxp系统结合腺病毒为载体携带Cre来敲除Rbl并过表达内耳前体细胞基因ISL1诱导新生及成年小鼠耳蜗毛细胞与支持细胞的增殖的研究。第一部分：5型腺病毒经中阶路径注射转染新生和成年小鼠耳蜗毛细胞和支持细胞的评价 [目的]为了研究5型腺病毒携带目的基因经新生和成年小鼠中阶入路导入转染内耳细胞的可行性,为以小鼠为动物模型的内耳基因治疗提供实验基础和解剖学依据. [方法]采用纳升级显微操作系统,经中阶显微注射携带增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein EGFP)基因的5型重组腺病毒或人工内淋巴液于各年龄段的小鼠耳蜗(p1,p4,p7,p12,p21,1-2M),内耳注射4天后取双侧耳蜗标本做基底膜铺片,观察EGFP表达情况。 [结果]p1、P4、P7组：术后第4天见耳蜗底圈、中圈支持细胞表达GFP。p12、p21、1-2M组：术后第4天可见耳蜗底圈、中圈支持细胞及部分内毛细胞表达EGFP。在人工内淋巴液和未注射耳,未见EGFP表达。 [结论]采用经中阶入路显微注射5型腺病毒携带目的基因导入新生鼠及成年鼠能够将目的基因成功转染至耳蜗组织并表达。第二部分：12种腺相关病毒经中阶路径注射转染新生和成年小鼠耳蜗毛细胞和支持细胞的比较 [目的]为了比较12种腺相关病毒携带目的基因经新生和成年小鼠中阶入路导入内耳的可行性,寻找新的更安全的载体,为以小鼠为动物模型的内耳基因治疗提供实验基础和解剖学依据。 [方法]采用经中阶显微注射12种腺相关病毒((AAV1、2、5、6、6.2、7、8、9、rh8、rh10、rh39、rh43))携带目的基因导入,将携带EGFP的腺相关病毒或人工内淋巴液导入小鼠耳蜗。其中新生鼠为p1/p2CD1小鼠,成年鼠为CBA/CAJ。新生鼠在内耳注射后1周、2周、3月,成年鼠在1周及3月后分别取双侧耳蜗标本做基底膜铺片及冰冻切片,观察EGFP表达情况。部分新生鼠组在术后3周行ABR检测。 [结果]新生鼠组：术后第1周、2周、3月可见支持细胞、毛细胞、螺旋韧带、螺旋神经节、前庭感觉细胞表达EGFP,GFP在术后1周表达较弱。术后ABR示听力约有0-15dB下降。其中AAV1和AAV2对支持细胞和毛细胞的感染效率相对较高。成年鼠组：术后1周、3月可见支持细胞、毛细胞、螺旋韧带、螺旋神经节、前庭感觉细胞表达EGFP。其中AAV1、AAV8和AAV9对支持细胞和毛细胞的感染效率相对较高。 [结论]采用经中阶显微注射腺相关病毒携带目的基因导入新生鼠及成年鼠能够将目的基因成功转染至耳蜗组织并表达,其中新生鼠组只有轻微的听力下降。新生鼠组可选择AAV1和AAV2作为载体,成年鼠组可选择AAV1、AAV8和AAV9。第三部分：敲除Rbl并过表达ISLl诱导小鼠耳蜗毛细胞与支持细胞增殖的体内研究 [目的]在体敲除Rbl并过表达ISL1诱导小鼠耳蜗毛细胞与支持细胞增殖的研究。 [方法]采用Er-Cre Rb loxp/loxp转基因鼠,体内模型中敲除Rbl基因,研究其对耳蜗毛细胞及支持细胞增殖的促进作用。以Er-Cre Rb loxp/loxp转基因鼠与ISL1转基因鼠杂交,获得Er-Cre Rb loxp/loxp ISL1或Rb loxp/loxp ISL1转基因鼠,在体敲除Rbl并过表达ISL-1,研究其对P1、P4、P5、P7、P12及P21耳蜗毛细胞及支持细胞增殖的促进作用。 [结果]在出生后的耳蜗毛细胞中特异性敲除Rbl基因,可见毛细胞和支持细胞在P4前能重新进入细胞周期,P5、P7、P12及P21未见毛细胞和支持细胞增殖。ISL1过表达在新生鼠和成年鼠中均未见毛细胞和支持细胞增殖,Rb1敲除联合ISL1过表达,也未见P4之后的毛细胞和支持细胞增殖。 [结论]Rb1基因敲除诱导耳蜗毛细胞和支持细胞增殖呈年龄相关性,Rb1敲除联合ISL1过表达也未能使得P4之后的毛细胞和支持细胞增殖。
[Abstract]:Lower vertebrates, such as birds, can regenerate for life after injury to the inner ear hair cells of the fish, and the mammalian cochlear hair cells have no spontaneous regeneration. Therefore, the sensorineural deafness caused by the loss of hair cells is still a major problem in clinical treatment. On the other hand, the hereditary deafness caused by hundreds of mutations in the gene is present. There is no treatment. Therefore, the development of hair cell regeneration and the development of the inner ear gene introduction have become the focus of hearing. The development of molecular biology has brought hope for the regeneration of hair cells. The gene therapy of the inner ear and the introduction of exogenous genes have brought new means for the treatment of deafness. Internal ear gene therapy is a common carrier. In order to verify the suitable vector for gene therapy, we use the newly developed nanoscale micromanipulation introduction system. We have studied the feasibility of the introduction of 12 different serotype AAV as gene carriers in a wide range. We found that different serotypes of AAV can be transferred in both newborn and adult rats. We also found that the injection of AAV in the newborn rats (P1/P2) to the inner ear can continue to express a long time and have little effect on hearing (0-15dB). This method can be used as a genetic deafness gene in the early development of the inner ear. Treatment.
The selective knockout of Rbl in the embryonic period of the inner ear hair cells and the progenitor cells of the support cells can produce a number of precursors that are far beyond the normal condition. These cells can further differentiate into hair cells and support cells. But after Rb1 knockout, the proliferation of hair cells and supporting cells is age-related, and the time cut is specific to the proliferation of cells. What is the function of Rbl knockout of the reentry function of sensory epithelial cells in the inner ear of different ages? Is it possible to combine other inner ear cells to allow more mature and adult mammalian hair cells to support cell proliferation? This study intends to transfect new and adult small adenoviruses through the midorder pathway of the 5 adenovirus. The evaluation of mouse cochlear hair cells and supporting cells; using Er-Cre Rb loxp/loxp system combined with adenovirus as a carrier to knock out Rbl and overexpress the gene ISL1 of the inner ear cells to induce the proliferation of cochlear hair cells and support cells in adult mice.
Part I: evaluation of the transfection of type 5 adenovirus into the cochlear hair cells and Sertoli cells of newborn and adult mice via middle route injection.
[Objective] to study the feasibility of transfection of the target gene of type 5 adenovirus carrying target gene into the inner ear cells through the middle order approach of new and adult mice, and to provide the experimental basis and anatomical basis for the internal ear gene therapy of mice as animal models.
[Methods] using the nanoscale micromanipulation system, the Enhanced Green Fluorescent Protein EGFP gene was injected into the cochlea (P1, P4, P7, p12, p21,1-2M) with the enhanced green fluorescent protein (Fluorescent Protein EGFP) gene, and the cochlear specimens were taken as basal membrane for 4 days after the inner ear injection. The expression of EGFP was observed.
[results]p1, P4, P7 group: the cochlear cochlea was seen on the fourth day after operation, and the middle ring support cells expressed GFP.p12. Group p21,1-2M: the cochlear cochlea was seen on the fourth day after operation. The middle ring support cells and some inner hair cells expressed EGFP. in the artificial lymph and uninjected ears, and no EGFP expression was found.
[Conclusion] the transfection of target genes into the cochlear tissues and expression of the target gene can be successfully transfected into the cochlear tissues by the introduction of the target gene carrying the target gene of the 5 type adenovirus.
The second part: comparison of 12 kinds of adeno-associated virus transfection into the cochlear hair cells and Sertoli cells of newborn and adult mice by middle route injection.
[Objective] to compare the feasibility of 12 adeno-related viruses carrying the target gene into the inner ear through the middle order approach of new and adult mice, to find a new and safer carrier and provide experimental basis and anatomical basis for the internal ear gene therapy of mice as animal model.
[Methods] 12 kinds of adeno-related viruses ((AAV1,2,5,6,6.2,7,8,9, rh8, rh10, rh39, rh43)) were introduced into the mouse cochlea with the adeno-related virus or artificial lymphatic fluid carrying EGFP. The newborn rats were p1/p2CD1 mice, and adult mice were injected with CBA/CAJ. newborn rats for 1 weeks, 2 weeks, March, and adult mice. After 1 weeks and March, bilateral cochlear specimens were taken for basement membrane preparation and frozen sections to observe the expression of EGFP. ABR was detected at 3 weeks after operation.
[results] first weeks, 2 weeks after operation, support cells, hair cells, spiral ligaments, spiral ganglion, vestibular sensory cells were expressed EGFP, and GFP was expressed weakly at 1 weeks after operation. 0-15dB decreased in ABR hearing after operation. Among them, the infection efficiency of AAV1 and AAV2 on support cells and capillary cells was relatively high. Adult rats: 1 weeks after operation, 3 Support cells, hair cells, spiral ligaments, spiral ganglion, vestibular sensory cells express EGFP. in this month, and AAV1, AAV8 and AAV9 are relatively efficient in the infection of support cells and hair cells.
[Conclusion] the target gene can be transfected into the cochlear tissue and expressed in the new rat and adult rat with the target gene of the midorder microinjection gland associated virus. The newborn rat group has only a slight hearing loss. The newborn rat group can choose AAV1 and AAV2 as the carrier, and the adult mouse group can choose AAV1, AAV8 and AAV9..
The third part: knocking out Rbl and overexpressing ISLl to induce the proliferation of cochlear hair cells and Sertoli cells in mice.
[Objective] in vivo knockdown of Rbl and overexpression of ISL1 induced the proliferation of cochlear hair cells and Sertoli cells in mice.
[Methods] the Er-Cre Rb loxp/loxp transgenic mice were used, and the Rbl gene was knocked out in the body model, and the promotion of the cochlear hair cells and the proliferation of the support cells was studied. The Er-Cre Rb loxp/loxp ISL1 or the transgenic mice were obtained by the hybridization of the Er-Cre Rb loxp/loxp transgenic mice and the ISL1 transgenic mice. To study the effects of P1, P4, P5, P7, P12 and P21 on the proliferation of hair cells and Sertoli cells in cochlea.
[results] the Rbl gene was specifically knocked out in the cochlear hair cells after birth. It was found that hair cells and supporting cells could reenter the cell cycle before P4. P5, P7, P12 and P21 did not see the proliferation of hair cells and support cells in the proliferation of hair cells and support cells in both newborn and adult rats. Rb1 knockout combined ISL1 overexpression, nor did Rb1 knock out. The proliferation of hair cells and supporting cells after P4 was seen.
[conclusion]Rb1 knockout induced cochlear hair cells and support cell proliferation is age-related. Rb1 knockout combined with ISL1 overexpression can not induce the proliferation of hair cells and support cells after P4.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R764

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