大鼠羊膜上皮細(xì)胞與膠原海綿復(fù)合體修復(fù)視神經(jīng)損傷的初步研究
發(fā)布時(shí)間：2019-07-05 17:05
【摘要】：目的:視神經(jīng)損傷后存活視網(wǎng)膜節(jié)細(xì)胞數(shù)量明顯減少,膠質(zhì)瘢痕形成及膠質(zhì)細(xì)胞釋放的抑制再生的各種分子,阻礙了再生軸突通過損傷區(qū)域,神經(jīng)再生困難。從組織工程的角度,我們將羊膜上皮細(xì)胞接種于膠原海綿上,體外構(gòu)建羊膜上皮細(xì)胞/膠原海綿復(fù)合體,移植入大鼠視神經(jīng)受損部位,初步觀察和探討復(fù)合體對(duì)損傷后視神經(jīng)修復(fù)的作用及相關(guān)機(jī)制。 方法:(1)取妊娠晚期SD大鼠的羊膜組織,貫序消化后進(jìn)行羊膜上皮細(xì)胞原代培養(yǎng),傳代細(xì)胞進(jìn)行免疫熒光細(xì)胞化學(xué)和定量PCR鑒定。(2)將傳代羊膜上皮細(xì)胞接種于膠原海綿上,體外共培養(yǎng)1周,行免疫熒光細(xì)胞化學(xué)、定量PCR鑒定。(3)用熒光染料標(biāo)記細(xì)胞、掃描電鏡、HE染色、CCK-8法等方法,檢測(cè)復(fù)合體上羊膜上皮細(xì)胞生長(zhǎng)及增殖情況。(4)將構(gòu)建的羊膜上皮細(xì)胞/膠原海綿復(fù)合體移植入成年雄性SD大鼠視神經(jīng)切斷傷模型中,設(shè)以下4組:正常對(duì)照組;損傷組,于眼球后約2 mm處用1ml注射器針尖將神經(jīng)外膜縱向切開,完全切除一段長(zhǎng)約0.5 mm的神經(jīng),致視神經(jīng)完全斷開,但保持視神經(jīng)外膜和血管完整,于視神經(jīng)缺損處注入10μl無血清培養(yǎng)基;空支架組是在損傷組的基礎(chǔ)上,于視神經(jīng)缺損處植入預(yù)培養(yǎng)1周的膠原海綿;實(shí)驗(yàn)組是在損傷組的基礎(chǔ)上,于視神經(jīng)缺損處移植入羊膜上皮細(xì)胞/膠原海綿復(fù)合體并注入10μl羊膜上皮細(xì)胞懸液(5×10~6個(gè)/ml)。(5)在部分羊膜上皮細(xì)胞/膠原海綿復(fù)合體移植組,用CM-Dil標(biāo)記支架上細(xì)胞的細(xì)胞膜,術(shù)后4周、8周經(jīng)心灌注固定,取視神經(jīng)作冰凍切片,觀察標(biāo)記細(xì)胞的存活和分布。(6)部分動(dòng)物取材前48 h,玻璃體注射CTB標(biāo)記視網(wǎng)膜節(jié)細(xì)胞,術(shù)后4周經(jīng)心灌注固定,取眼球視網(wǎng)膜鋪片,計(jì)數(shù)CTB標(biāo)記細(xì)胞;部分動(dòng)物術(shù)后4周經(jīng)心灌注固定,取眼球視網(wǎng)膜鋪片,尼氏染色計(jì)數(shù)視網(wǎng)膜節(jié)細(xì)胞;部分動(dòng)物術(shù)后4周、8周取視神經(jīng)眶內(nèi)段HE染色觀察組織結(jié)構(gòu)和細(xì)胞密度變化,免疫組織化學(xué)染色法顯示GAP-43表達(dá)。 結(jié)果: (1)體外成功培養(yǎng)獲得大鼠羊膜上皮細(xì)胞,接種于膠原海綿,免疫熒光細(xì)胞化學(xué)染色鑒定后顯示,接種膠原海綿前后,羊膜上皮細(xì)胞均能表達(dá)上皮細(xì)胞特異性標(biāo)志物CK-19、神經(jīng)干細(xì)胞標(biāo)志物Nestin、以及細(xì)胞多能性標(biāo)志分子Oct-4、Nanog等。(2)定量PCR檢測(cè),結(jié)果顯示羊膜上皮細(xì)胞接種膠原海綿前后均有CK-19、Nestin、Oct-4、Nanog、bFGF的mRNA表達(dá),且細(xì)胞接種膠原海綿后Nestin的mRNA表達(dá)顯著上調(diào)。(3)經(jīng)熒光標(biāo)記、掃描電鏡、HE染色、CKK-8法檢測(cè),結(jié)果顯示羊膜上皮細(xì)胞能較好地粘附于膠原海綿上生長(zhǎng),膠原海綿能促進(jìn)羊膜上皮細(xì)胞增殖。(4)將用CM-Dil標(biāo)記細(xì)胞的復(fù)合體移植入大鼠視神經(jīng)切斷傷模型后,術(shù)后4周、8周取視神經(jīng)行冰凍切片,可觀察到損傷區(qū)有標(biāo)記的細(xì)胞存活,能向受損視神經(jīng)近側(cè)段和遠(yuǎn)側(cè)段遷移,并可沿神經(jīng)外膜向兩側(cè)遷移。(5)損傷后4周,CTB及尼氏染色視網(wǎng)膜節(jié)細(xì)胞的計(jì)數(shù)結(jié)果顯示,各組節(jié)細(xì)胞密度較正常組明顯降低,細(xì)胞復(fù)合體組節(jié)細(xì)胞密度較損傷組、空支架組增加;空支架組的節(jié)細(xì)胞密度較損傷組無明顯差異。(6)HE染色視神經(jīng)顯示,膠原海綿移植體內(nèi)后,能與斷端神經(jīng)組織相融合,術(shù)后8周基本降解。視神經(jīng)損傷區(qū)有大量細(xì)胞存在,呈不規(guī)則條索狀分布并向兩端延伸。視神經(jīng)損傷后遠(yuǎn)側(cè)段細(xì)胞核明顯變小,而復(fù)合體移植組的胞核與正常對(duì)照組大小相似。傷后4周、8周計(jì)數(shù)遠(yuǎn)側(cè)段細(xì)胞結(jié)果顯示,各組細(xì)胞數(shù)量均高于正常組,空支架組較損傷組細(xì)胞數(shù)量無增加,細(xì)胞復(fù)合體組細(xì)胞數(shù)量增加明顯高于損傷組及空支架組。(7)免疫組織化學(xué)法對(duì)視神經(jīng)GAP-43染色后顯示,損傷組、空支架組中GAP-43在損傷區(qū)僅有少量表達(dá),遠(yuǎn)側(cè)段未見表達(dá);復(fù)合體移植后損傷區(qū)GAP-43表達(dá)明顯增多,可見少量GAP-43陽性、類似再生軸突樣結(jié)構(gòu),由傷區(qū)伸入至遠(yuǎn)側(cè)段,8周時(shí)能較4周達(dá)到更遠(yuǎn)處。 結(jié)論:(1)羊膜上皮細(xì)胞與膠原海綿組織相容性好,膠原海綿能促進(jìn)羊膜上皮細(xì)胞的增殖,促進(jìn)羊膜上皮細(xì)胞向神經(jīng)干細(xì)胞方向分化,復(fù)合體活性較好;(2)復(fù)合體移植受損視神經(jīng)后,部分復(fù)合體細(xì)胞能在損傷部位存活至少8周并向損傷區(qū)兩側(cè)遷移,能在一定程度上保護(hù)視網(wǎng)膜節(jié)細(xì)胞和視神經(jīng)的神經(jīng)膠質(zhì)細(xì)胞,增強(qiáng)視神經(jīng)再生軸突的生長(zhǎng)活力,特別是能促使少量再生軸突通過損傷部位進(jìn)入到遠(yuǎn)側(cè)段神經(jīng),表明所構(gòu)建的復(fù)合體能改善視神經(jīng)再生微環(huán)境、促進(jìn)軸突再生。
[Abstract]:Objective: The number of viable retinal ganglion cells after optic nerve injury was significantly reduced, and the formation of glial scar and the release of glial cells inhibited the regeneration of various molecules, which prevented the regeneration of axons from the area of injury and the difficulty of nerve regeneration. From the angle of tissue engineering, the amniotic epithelial cells were inoculated on the collagen sponge, the amniotic epithelial cells/ collagen sponge complex was constructed in vitro, the damaged part of the optic nerve of the rat was transplanted, and the effect of the complex on the optic nerve repair after injury and the related mechanism were observed and discussed. Methods: (1) The amniotic epithelial cells of SD rats were cultured in the late stage of pregnancy, and the primary culture and passage of the cells were carried out by immunofluorescence cell chemistry and quantitative PCR. and (2) inoculating the subcultured amniotic epithelial cells on the collagen sponge, co-culturing for 1 week in vitro, carrying out fluorescent cell chemistry and quantitative PCR in vitro, (3) detecting the growth and proliferation of amniotic epithelial cells on the complex by means of fluorescent dye-labeled cells, scanning electron microscopy (SEM), HE staining, and CCK-8 method. Cases. (4) The constructed amniotic epithelial cell/ collagen sponge complex was transplanted into the optic nerve cut-out model of adult male SD rats, and the following four groups were set: normal control group; injury group;1 ml of syringe needle tip was used to machine the outer membrane of the nerve in the longitudinal direction at about 2 mm after the eyeball. Incision, complete removal of a length of nerve with a length of about 0.5 mm, complete disconnection of the optic nerve, but keep the outer membrane of the optic nerve and the blood vessel intact, inject 10. m u.l of serum-free medium at the optic nerve defect; the empty stent group is on the basis of the damaged group, and the pre-cultured 1-week collagen is implanted in the optic nerve defect. Sponge; the experimental group was transplanted into the amniotic epithelial cell/ collagen sponge complex at the optic nerve defect and injected with 10 & mu; l of amniotic epithelial cell suspension (5-10-6/ m) on the basis of the injury group. (1). (5) In the transplantation group of part of the amniotic epithelial cell/ collagen sponge complex, the cell membrane of the cell was marked with CM-Dil, the cell membrane of the cell was labeled with CM-Dil, the cell membrane of the cell was fixed at 4 and 8 weeks after operation, the optic nerve was taken as a frozen section, and the survival of the labeled cells was observed. Distribution. (6) The vitreoretinal ganglion cells were labeled with CTB in vitreoretinal injection for 48 hours before and after 4 weeks of operation, and the retina of the eye was set to count the CTB-labeled cells. The 4-week post-operation of some animals was fixed by heart-perfusion, and the retina of the eye was taken. The changes of tissue structure and cell density were observed at 4 weeks and 8 weeks after operation, and the changes of tissue structure and cell density were observed in 8 weeks after operation, and GAP-43 was shown by immunohistochemical staining. Results: (1) In vitro, the amniotic epithelial cells of the rat were successfully cultured and inoculated in the collagen sponge. After the chemical staining and identification of the immunofluorescence cells, the epithelial cell-specific markers CK-19 and the neural stem cell marker N could be expressed in the amniotic epithelial cells before and after the inoculation of the collagen sponge. estin, and cell multi-functional marker molecule Oct-4, N The expression of CK-19, Nestin, Oct-4, Nanog, and bFGF in the amniotic epithelial cells was detected by quantitative PCR. The results showed that the amniotic epithelial cells could be better adhered to the collagen sponge and the collagen sponge could promote the amniotic membrane. (4) After transplantation of the complex of the CM-Dil-labeled cells into the rat optic nerve-cutting injury model, the optic nerve line frozen section was taken at 4 and 8 weeks after the operation, and the labeled cells in the damaged area were observed to survive, and the injured optic nerve proximal section and the damaged optic nerve were observed. The distal segment is migrated and can be along the outer membrane of the nerve The results showed that the cell density of the cell complex was lower than that in the normal group, the cell density of the cell complex group was lower than that of the group, the empty stent group was increased, and the cell density in the empty stent group was lower than that in the group. There was no significant difference. (6) HE staining the optic nerve showed that after the collagen sponge was transplanted into the body, it could be fused with the end-end nerve tissue, and the operation was 8. The optic nerve injury area has a large number of cells, which are distributed in the form of irregular strips. The nucleus of the distal segment was significantly smaller after the optic nerve injury, and the nucleus of the complex transplantation group and the normal control. The results showed that the number of cells in the group was higher than that in the normal group, and the number of cells in the cell complex group was significantly higher than that of the injured group. (7) The GAP-43 expression of GAP-43 was only expressed in the injured area after GAP-43 was stained by immunocytochemical method. The expression of GAP-43 in the damaged area was not seen in the distal segment. The expression of GAP-43 in the damaged area after the complex transplantation was significantly increased, and a small amount of GAP-43 was found to be positive and similar to the regeneration. The axon-like structure, which extends into the distal section from the wound area, can be more than 4 weeks at 8 weeks Conclusion: (1) The collagen sponge has good compatibility with the collagen sponge, and the collagen sponge can promote the proliferation of the amniotic epithelial cells and promote the differentiation of the amniotic epithelial cells in the direction of the neural stem cells, and the activity of the complex is better; (2) the complex after the damaged optic nerve is transplanted, the part of the complex cells can survive for at least 8 weeks at the damaged site and migrate to the two sides of the damaged area, so that the retinal ganglion cells and the glial cells of the optic nerve can be protected to a certain extent, the optic nerve is enhanced, The growth vigor of the axons, in particular, can cause a small amount of regenerated axons to enter the nerve of the distal section through the lesion site, indicating that the constructed composite can improve the micro-ring of the optic nerve regeneration.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R774.6
本文編號(hào)：2510681
[Abstract]:Objective: The number of viable retinal ganglion cells after optic nerve injury was significantly reduced, and the formation of glial scar and the release of glial cells inhibited the regeneration of various molecules, which prevented the regeneration of axons from the area of injury and the difficulty of nerve regeneration. From the angle of tissue engineering, the amniotic epithelial cells were inoculated on the collagen sponge, the amniotic epithelial cells/ collagen sponge complex was constructed in vitro, the damaged part of the optic nerve of the rat was transplanted, and the effect of the complex on the optic nerve repair after injury and the related mechanism were observed and discussed. Methods: (1) The amniotic epithelial cells of SD rats were cultured in the late stage of pregnancy, and the primary culture and passage of the cells were carried out by immunofluorescence cell chemistry and quantitative PCR. and (2) inoculating the subcultured amniotic epithelial cells on the collagen sponge, co-culturing for 1 week in vitro, carrying out fluorescent cell chemistry and quantitative PCR in vitro, (3) detecting the growth and proliferation of amniotic epithelial cells on the complex by means of fluorescent dye-labeled cells, scanning electron microscopy (SEM), HE staining, and CCK-8 method. Cases. (4) The constructed amniotic epithelial cell/ collagen sponge complex was transplanted into the optic nerve cut-out model of adult male SD rats, and the following four groups were set: normal control group; injury group;1 ml of syringe needle tip was used to machine the outer membrane of the nerve in the longitudinal direction at about 2 mm after the eyeball. Incision, complete removal of a length of nerve with a length of about 0.5 mm, complete disconnection of the optic nerve, but keep the outer membrane of the optic nerve and the blood vessel intact, inject 10. m u.l of serum-free medium at the optic nerve defect; the empty stent group is on the basis of the damaged group, and the pre-cultured 1-week collagen is implanted in the optic nerve defect. Sponge; the experimental group was transplanted into the amniotic epithelial cell/ collagen sponge complex at the optic nerve defect and injected with 10 & mu; l of amniotic epithelial cell suspension (5-10-6/ m) on the basis of the injury group. (1). (5) In the transplantation group of part of the amniotic epithelial cell/ collagen sponge complex, the cell membrane of the cell was marked with CM-Dil, the cell membrane of the cell was labeled with CM-Dil, the cell membrane of the cell was fixed at 4 and 8 weeks after operation, the optic nerve was taken as a frozen section, and the survival of the labeled cells was observed. Distribution. (6) The vitreoretinal ganglion cells were labeled with CTB in vitreoretinal injection for 48 hours before and after 4 weeks of operation, and the retina of the eye was set to count the CTB-labeled cells. The 4-week post-operation of some animals was fixed by heart-perfusion, and the retina of the eye was taken. The changes of tissue structure and cell density were observed at 4 weeks and 8 weeks after operation, and the changes of tissue structure and cell density were observed in 8 weeks after operation, and GAP-43 was shown by immunohistochemical staining. Results: (1) In vitro, the amniotic epithelial cells of the rat were successfully cultured and inoculated in the collagen sponge. After the chemical staining and identification of the immunofluorescence cells, the epithelial cell-specific markers CK-19 and the neural stem cell marker N could be expressed in the amniotic epithelial cells before and after the inoculation of the collagen sponge. estin, and cell multi-functional marker molecule Oct-4, N The expression of CK-19, Nestin, Oct-4, Nanog, and bFGF in the amniotic epithelial cells was detected by quantitative PCR. The results showed that the amniotic epithelial cells could be better adhered to the collagen sponge and the collagen sponge could promote the amniotic membrane. (4) After transplantation of the complex of the CM-Dil-labeled cells into the rat optic nerve-cutting injury model, the optic nerve line frozen section was taken at 4 and 8 weeks after the operation, and the labeled cells in the damaged area were observed to survive, and the injured optic nerve proximal section and the damaged optic nerve were observed. The distal segment is migrated and can be along the outer membrane of the nerve The results showed that the cell density of the cell complex was lower than that in the normal group, the cell density of the cell complex group was lower than that of the group, the empty stent group was increased, and the cell density in the empty stent group was lower than that in the group. There was no significant difference. (6) HE staining the optic nerve showed that after the collagen sponge was transplanted into the body, it could be fused with the end-end nerve tissue, and the operation was 8. The optic nerve injury area has a large number of cells, which are distributed in the form of irregular strips. The nucleus of the distal segment was significantly smaller after the optic nerve injury, and the nucleus of the complex transplantation group and the normal control. The results showed that the number of cells in the group was higher than that in the normal group, and the number of cells in the cell complex group was significantly higher than that of the injured group. (7) The GAP-43 expression of GAP-43 was only expressed in the injured area after GAP-43 was stained by immunocytochemical method. The expression of GAP-43 in the damaged area was not seen in the distal segment. The expression of GAP-43 in the damaged area after the complex transplantation was significantly increased, and a small amount of GAP-43 was found to be positive and similar to the regeneration. The axon-like structure, which extends into the distal section from the wound area, can be more than 4 weeks at 8 weeks Conclusion: (1) The collagen sponge has good compatibility with the collagen sponge, and the collagen sponge can promote the proliferation of the amniotic epithelial cells and promote the differentiation of the amniotic epithelial cells in the direction of the neural stem cells, and the activity of the complex is better; (2) the complex after the damaged optic nerve is transplanted, the part of the complex cells can survive for at least 8 weeks at the damaged site and migrate to the two sides of the damaged area, so that the retinal ganglion cells and the glial cells of the optic nerve can be protected to a certain extent, the optic nerve is enhanced, The growth vigor of the axons, in particular, can cause a small amount of regenerated axons to enter the nerve of the distal section through the lesion site, indicating that the constructed composite can improve the micro-ring of the optic nerve regeneration.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R774.6
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
1 呂立權(quán);樓美清;董艷;蔡如玨;胡國(guó)漢;駱純;侯立軍;盧亦成;;視神經(jīng)慢性受壓后視神經(jīng)膠質(zhì)細(xì)胞的病理變化[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2009年06期
2 黃蔚,惠延年,呂榮,王琳,馬吉獻(xiàn);視神經(jīng)擠壓傷后星形膠質(zhì)細(xì)胞和軸突反應(yīng)的免疫組織化學(xué)研究(英文)[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2001年06期
3 關(guān)靜,武繼民;膠原蛋白的醫(yī)療應(yīng)用[J];軍事醫(yī)學(xué)科學(xué)院院刊;1997年04期
4 鄒海燕,葉春婷,李斯明,彭燕豪,葉惠貞;bFGF膠原海綿的制備及其組織相容性評(píng)價(jià)[J];生物醫(yī)學(xué)工程與臨床;2003年01期
5 藺海燕;許家軍;劉芳;郭金萍;;視神經(jīng)再生微環(huán)境的調(diào)控[J];神經(jīng)解剖學(xué)雜志;2006年04期
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