發(fā)布時(shí)間：2018-03-04 01:13

  本文選題：脊髓康　切入點(diǎn)：小膠質(zhì)細(xì)胞　出處：《南京中醫(yī)藥大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：脊髓損傷(Spinal cord injury,SCI)是一種嚴(yán)重致殘性疾病,其引起的殘疾無(wú)論對(duì)患者、對(duì)家庭或?qū)ι鐣?huì)都是一種最嚴(yán)重的災(zāi)難,且痛苦和負(fù)擔(dān)往往伴隨終身。目前基礎(chǔ)及臨床研究認(rèn)為,SCI后神經(jīng)元的繼發(fā)性損傷是可能被逆轉(zhuǎn)的,這為SCI治療提供了可研究方向。小膠質(zhì)細(xì)胞是駐留于中樞神經(jīng)系統(tǒng)(Central nervous system,CNS)內(nèi)的免疫細(xì)胞,其為CNS的免疫與防御系統(tǒng)的重要組成部分,能夠保護(hù)CNS免受各種病理因子的侵害,但其有時(shí)亦會(huì)導(dǎo)致CNS相關(guān)病變的進(jìn)一步惡化。當(dāng)中風(fēng)、腦外傷、SCI等神經(jīng)病變發(fā)生后,小膠質(zhì)細(xì)胞在對(duì)損傷局部神經(jīng)元表現(xiàn)出破壞、修復(fù)的雙重作用。一方面,小膠質(zhì)細(xì)胞可做出迅速的免疫應(yīng)答,定向遷移至病變部位后釋放大量促炎因子啟動(dòng)炎性反應(yīng),過(guò)度的、反復(fù)的炎癥反應(yīng)及有害代謝產(chǎn)物對(duì)神經(jīng)系統(tǒng)造成嚴(yán)重的損害,導(dǎo)致大量神經(jīng)元的繼發(fā)性壞死及凋亡,影響神經(jīng)元的修復(fù)、再生;另一方面,小膠質(zhì)細(xì)胞遷移至損傷部位后,其吞噬作用可通過(guò)清除凋亡神經(jīng)元,減少細(xì)胞因子、神經(jīng)毒性物質(zhì)等釋放,進(jìn)而抑制過(guò)度的免疫炎性反應(yīng),協(xié)調(diào)、促進(jìn)神經(jīng)元網(wǎng)絡(luò)狀結(jié)構(gòu)的重建及功能的恢復(fù)。故而小膠質(zhì)細(xì)胞在CNS生理病理變化中有著重要意義。"脊髓康"是筆者導(dǎo)師臨床經(jīng)驗(yàn)方,臨床實(shí)踐及研究證明該方有著良好的臨床療效�；诖朔�,我們前期系統(tǒng)開(kāi)展了多項(xiàng)"脊髓康"干預(yù)SCI的基礎(chǔ)性研究,結(jié)果表明,"脊髓康"對(duì)SCI后免疫應(yīng)答有著重要的調(diào)節(jié)作用,其能有效促進(jìn)損傷神經(jīng)元的修復(fù)再生。目前,中醫(yī)藥對(duì)小膠質(zhì)細(xì)胞吞噬作用的研究較少,已有研究主要局限于部分中藥能夠抑制小膠質(zhì)細(xì)胞活化所致的過(guò)度免疫炎性反應(yīng)以降低神經(jīng)二次損傷的方面,基于此,本研究欲從小膠質(zhì)細(xì)胞吞噬作用角度,探討"脊髓康"有效干預(yù)SCI的潛在機(jī)制,為SCI的臨床治療提供一定科學(xué)依據(jù)。本文第一部分采用慢病毒轉(zhuǎn)染綠色熒光蛋白基因標(biāo)記小膠質(zhì)細(xì)胞株BV2,核染色標(biāo)記死亡神經(jīng)元的方法,建立了一個(gè)可動(dòng)態(tài)觀察BV2吞噬神經(jīng)元碎片的體系,研究并證實(shí)了"脊髓康"可促進(jìn)BV2對(duì)神經(jīng)元碎片的吞噬作用;我們認(rèn)為,中樞神經(jīng)系統(tǒng)是一個(gè)由多種膠質(zhì)細(xì)胞及神經(jīng)元構(gòu)成的復(fù)雜體系,SCI的發(fā)生必然不是單一系統(tǒng)或某一細(xì)胞群的病變,而是多因素的"整體病變",故在第一部分的研究基礎(chǔ)上,我們提取胎鼠的部分神經(jīng)組織,未經(jīng)特殊篩選將其接種于體外環(huán)境中培養(yǎng),初步、簡(jiǎn)易的模擬了一個(gè)多因素、立體式、整體性的混雜細(xì)胞體系,在誘導(dǎo)混雜細(xì)胞體系中神經(jīng)元凋亡后予"脊髓康"干預(yù),再次證實(shí)了"脊髓康"對(duì)模擬的"自然混雜體系"中小膠質(zhì)細(xì)胞吞噬功能的促進(jìn)作用,體現(xiàn)了中醫(yī)藥"整體"干預(yù)的理念;上述兩部分實(shí)驗(yàn)表明,"脊髓康"能有效調(diào)控小膠質(zhì)細(xì)胞的吞噬功能,那么該作用到底是否與促神經(jīng)修復(fù)有相關(guān)性?基于這些問(wèn)題,我們開(kāi)展了第三部分實(shí)驗(yàn),即驗(yàn)證"脊髓康"是否能通過(guò)調(diào)控小膠質(zhì)細(xì)胞吞噬作用影響神經(jīng)元修復(fù)再生,在此實(shí)驗(yàn)中,我們成功分離、提純了原代神經(jīng)元、原代小膠質(zhì)細(xì)胞,建立小膠質(zhì)細(xì)胞/損傷神經(jīng)元共培養(yǎng)體系,"脊髓康"含藥血清間接干預(yù)共培養(yǎng)體系,而后以免疫熒光雙重標(biāo)記法觀察混培養(yǎng)24h后小膠質(zhì)細(xì)胞對(duì)神經(jīng)元碎片的吞噬作用及96h后損傷神經(jīng)元突起生長(zhǎng)情況,我們發(fā)現(xiàn)"脊髓康"促進(jìn)小膠質(zhì)細(xì)胞清理凋亡神經(jīng)元碎片的同時(shí),存活神經(jīng)元的修復(fù)再生能力得到了明顯的提升。綜上,我們得出:中藥復(fù)方"脊髓康"能夠有效促進(jìn)小膠質(zhì)細(xì)胞吞噬神經(jīng)元碎片作用,可能以此改善局部微環(huán)境,促進(jìn)損傷神經(jīng)元的修復(fù)再生。第一部分"脊髓康"對(duì)小膠質(zhì)細(xì)胞吞噬神經(jīng)元碎片的影響目的:觀察中藥復(fù)方"脊髓康"對(duì)小膠質(zhì)細(xì)胞吞噬神經(jīng)元碎片的影響。方法:制備"脊髓康"含藥血清,分離、培養(yǎng)、鑒定原代神經(jīng)元,慢病毒轉(zhuǎn)染綠色熒光蛋白基因標(biāo)記小膠質(zhì)細(xì)胞株BV2,核染色標(biāo)記神經(jīng)元,熒光顯微鏡下觀察含藥血清干預(yù)后小膠質(zhì)細(xì)胞對(duì)神經(jīng)元碎片的吞噬作用。結(jié)果:在吞噬百分率方面,"脊髓康"中、高劑量組均高于空白組(P0.05),與脂多糖組對(duì)比差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);在吞噬指數(shù)方面,"脊髓康"低、中、高劑量組均高于空白組(P0.05),中、高劑量組與脂多糖組對(duì)比差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:中藥復(fù)方"脊髓康"能調(diào)控BV2對(duì)神經(jīng)元碎片的吞噬作用,其促進(jìn)損傷神經(jīng)元軸突再生的機(jī)制可能與此相關(guān)。此外,本研究為中醫(yī)藥調(diào)控小膠質(zhì)細(xì)胞干預(yù)CNS相關(guān)疾病的研究提供了新的思路及方法。第二部分"脊髓康"對(duì)自然混雜體系中原代小膠質(zhì)細(xì)胞吞噬作用的影響目的:觀察中藥復(fù)方"脊髓康"對(duì)自然混雜體系中原代小膠質(zhì)細(xì)胞吞噬神經(jīng)元碎片的影響。方法:制備"脊髓康"含藥血清;分離、培養(yǎng)原代神經(jīng)細(xì)胞,建立混雜細(xì)胞體系并予鑒定;以谷氨酸誘導(dǎo)混雜體系中神經(jīng)元損傷,以"脊髓康"含藥血清干預(yù)混雜體系;以一抗β3-tubulin、二抗Alexa 594標(biāo)記神經(jīng)元骨架及碎片,以一抗CD11b、二抗Alexa 488標(biāo)記小膠質(zhì)細(xì)胞相關(guān)膜蛋白,觀察"脊髓康"干預(yù)后混雜體系中小膠質(zhì)細(xì)胞對(duì)神經(jīng)元碎片的吞噬作用。結(jié)果:在吞噬指數(shù)方面,"脊髓康"含藥血清低、中、高劑量組均高于空白組(P0.05),中、高劑量組較LPS組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);在吞噬百分率方面,中、高劑量組均高于空白組(P0.05),中劑量組低于LPS組(P0.05)。結(jié)論:抗SCI有效方"脊髓康"可調(diào)控小膠質(zhì)細(xì)胞清理神經(jīng)元碎片的作用,改善局部微環(huán)境,促進(jìn)損傷神經(jīng)元的修復(fù)再生。第三部分"脊髓康"對(duì)共培養(yǎng)體系中小膠質(zhì)細(xì)胞吞噬及損傷神經(jīng)元再生的影響目的:觀察中藥復(fù)方"脊髓康"對(duì)共培養(yǎng)體系中小膠質(zhì)細(xì)胞吞噬及損傷神經(jīng)元修復(fù)再生的影響。方法:制備"脊髓康"含藥血清,分離、鑒定原代海馬神經(jīng)元、原代小膠質(zhì)細(xì)胞,谷氨酸誘導(dǎo)神經(jīng)元損傷,含藥血清預(yù)處理小膠質(zhì)細(xì)胞,建立小膠質(zhì)細(xì)胞/損傷神經(jīng)元共培養(yǎng)體系,采用免疫熒光雙重標(biāo)記法觀察混培養(yǎng)24h后小膠質(zhì)細(xì)胞對(duì)神經(jīng)元碎片的吞噬作用及96h后損傷神經(jīng)元突起生長(zhǎng)情況。結(jié)果:混培養(yǎng)24h后,"脊髓康"含藥血清中、高劑量組在吞噬指數(shù)及吞噬百分率方面均高于空白組(P0.05),中劑量組低于LPS組(P0.05),高劑量組則與LPS組無(wú)顯著差異(P0.05)。混培養(yǎng)96h后,"脊髓康"含藥血清中、高劑量組一級(jí)突起數(shù)量多于空白組(P0.05),與LPS組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。"脊髓康"中、高劑量組平均突起較空白組長(zhǎng)(P0.05),與LPS對(duì)比,"脊髓康"中劑量組平均神經(jīng)突起較短(P0.05),而高劑量組則平均突起較長(zhǎng)(P0.05)。結(jié)論:"脊髓康"通過(guò)調(diào)控小膠質(zhì)細(xì)胞吞噬功能促進(jìn)損傷神經(jīng)元的修復(fù)與再生,這可能與其促進(jìn)凋亡物質(zhì)清理后,于局部微環(huán)境建立一個(gè)無(wú)形的保護(hù)屏障有關(guān),但其詳細(xì)機(jī)制仍待進(jìn)一步研究。綜上所述,本研究工作的主要?jiǎng)?chuàng)新之處在于:1首次從調(diào)控小膠質(zhì)細(xì)胞吞噬作用的角度對(duì)中醫(yī)藥干預(yù)SCI進(jìn)行了研究,為"脊髓康"溫腎通督抗SCI作用做出了新的科學(xué)闡釋。2自行研究、建立了多種能準(zhǔn)確、有效以及動(dòng)態(tài)觀察小膠質(zhì)細(xì)胞吞噬神經(jīng)元碎片的方法,驗(yàn)證了"脊髓康"對(duì)小膠質(zhì)細(xì)胞吞噬的調(diào)控作用,一定程度上明確了該作用與神經(jīng)修復(fù)再生的相互關(guān)系。3基于中醫(yī)藥干預(yù)"整體"理念,及SCI整體病變的思考,體外建立了一個(gè)多因素、整體性的混雜體系,并進(jìn)行了初步的研究。
[Abstract]:Spinal cord injury (Spinal cord, injury, SCI) is a serious disabling disease, its cause of disability to the patient, the family or are one of the most serious disaster to the society, and is often accompanied by pain and burden of life. The basic and clinical research that SCI induced secondary injury of neurons is likely to be reversed, this provides the research direction for the treatment of SCI. Microglia are resident in the central nervous system (Central nervous system, CNS) in immune cells, an important part of the immune defense system and CNS, can protect CNS from various pathological factors of infringement, but its sometimes will also lead to further the deterioration of diseases related to CNS. When the stroke, brain trauma, etc. SCI neuropathy, microglia in the injury of local neurons showed destruction of the dual role of repair. On the one hand, microglial cells can make rapid free Immune response, migrate to the lesion after the release of a large number of proinflammatory cytokines that initiate the inflammatory reaction, excessive, repeated inflammation and harmful metabolites cause serious damage to the nervous system, resulting in a large number of secondary neuronal necrosis and apoptosis of neurons, repair, regeneration; on the other hand, microglial cells migrate to after the injury, the phagocytosis by clearance of apoptotic neurons, reduced cytokine release, neurotoxic substances, and then inhibit the excessive inflammatory reaction, coordination, promote reconstruction and function of neural network structure recovery. Therefore microglia plays an important role in the physiological and pathological changes in CNS Jisuikang. "Is the author of my tutor's clinical experience, clinical practice and research show that the party has a good clinical effect. Based on this, we carried out a number of pre system based" Jisuikang "intervention SCI Research results show that "Jisuikang" plays an important role in the regulation of immune response after SCI, which can effectively promote the repair and regeneration of injured neurons. At present, Chinese medicine phagocytosis less research on microglia, the existing research is mainly limited to excessive immune inflammatory reaction of some traditional Chinese medicine can inhibit the activation of microglia in order to reduce two times caused by nerve injury, based on this, this research intends to small glial cell phagocytosis angle, explore potential mechanisms of Jisuikang "effective intervention of SCI, to provide a scientific basis for clinical treatment of SCI. In the first part of this paper using lentiviral marker transfected with green fluorescent protein gene of microglial cell line BV2. Nuclear staining of dead neurons, we set up a dynamic observation of BV2 neuron debris phagocytic system, and the results confirmed the" Jisuikang "can promote BV2 on neuronal debris swallow Apoptosis; we believe that the central nervous system is a complex system composed of a variety of glial cells and neurons, the occurrence of SCI is not necessarily a single system or a group of cells of the lesion, but many factors ", so the overall lesions based on the first part of the study, we extracted nerve tissue of fetal rats without special screening, were inoculated with in vitro culture environment, preliminary, simple simulation of a multi factor, three-dimensional, hybrid cell system integrity, in the induction of neuronal apoptosis after cell hybrid system to" Jisuikang "intervention, once again confirmed the role of simulated" Jisuikang "the natural hybrid system" the phagocytic function of microglia, reflects the traditional Chinese medicine "whole" intervention idea; show that the two part of the experiment, "Jisuikang" can effectively control the phagocytic function of microglia, the role of promoting and whether There is a correlation between the nerve repair? Based on these questions, we conducted third experiments, which verify the "Jisuikang" whether through the regulation of microglial phagocytosis of neuron regeneration, in this experiment, we successfully isolated and purified primary neurons, primary microglia, establishment of microglia / total neuronal injury training system, "Jisuikang" medicated serum indirect intervention co culture system, and then by double immunofluorescent staining method to observe the mixed culture of 24h microglia on neuronal debris phagocytosis and 96h after injury of neurite growth, we find that "Jisuikang" to promote microglia clear neuronal apoptosis fragments at the same time, the ability of regeneration repair of surviving neurons has been significantly improved. In summary, we conclude that: the traditional Chinese medicine "Jisuikang" can effectively promote microglia phagocytosis of neuronal debris May, in order to improve the local micro environment, promote the repair and regeneration of injured neurons. The first part "Jisuikang" phagocytosis of neurons debris on the microglial cells Objective: To observe the effect of traditional Chinese medicine "Jisuikang" phagocytic debris on microglia neurons. Methods: the preparation of "Jisuikang" serum, isolation, culture primary identification of neurons, labeled, lentiviral transfection of green fluorescent protein gene on BV2 cell line, nuclear staining labeled neurons, observe serum stem phagocytosis of microglia on neuronal prognosis fragments under fluorescence microscope. Results: the percentage of phagocytosis, "Jisuikang" in high dose group were higher than those of control group (P0.05), there was no significant difference compared with LPS group (P0.05); the phagocytic index, "Jisuikang" low, high dose group were higher than those of control group (P0.05), and the high dose group and LPS group contrast There was no statistically significant difference (P0.05). Conclusion: the traditional Chinese medicine "Jisuikang" phagocytosis regulation of BV2 on neuronal debris, the damage mechanism of promoting axonal regeneration may be related to this. In addition, provides new ideas and methods of this research for the regulation of Chinese medicine intervention of microglia CNS related diseases the second part "Jisuikang. Effect of natural hybrid system primary microglial phagocytosis Objective: To observe the effect of traditional Chinese medicine" Jisuikang "of natural hybrid system primary microglial phagocytosis of neuronal debris. Methods: the preparation of" Jisuikang "containing serum; separation of primary cultured nerve cells, hybrid and to establish the system of cell identification; neuronal damage induced by glutamate in the hybrid system, hybrid system to intervention" Jisuikang "to a drug containing serum; anti beta 3-tubulin, two anti Alexa 594 labeled neurons and fragments of skeleton In two, anti CD11b, anti Alexa 488 labeled microglia associated membrane protein, observe the "Jisuikang" stem phagocytosis prognosis hybrid system of microglia on neuronal debris. Results: the phagocytic index, "Jisuikang" serum containing low and high dose group were higher than those of control group (P0.05) in the high dose group, the difference was not statistically significant compared with LPS group (P0.05); the phagocytic percentage, in high dose group were higher than those of control group (P0.05), middle dose group was lower than that of LPS group (P0.05). Conclusion: Anti SCI effective "Jisuikang" regulation of microglia neuron debris cleanup the role, improve the local micro environment, promote the repair and regeneration of injured neurons. In the third part, "Jisuikang" on the effect of co culture system of microglial phagocytosis and injury neuron regeneration Objective: To observe the traditional Chinese medicine "Jisuikang" system of microglial phagocytosis and damage God co culture The influence of element repair and regeneration. Methods: the preparation of "Jisuikang" serum, separation, identification of primary hippocampal neurons, primary microglia neuron injury induced by glutamate, serum pretreatment of microglia, establishment of microglia / damaged neurons co culture system using double immunofluorescent staining method to observe the mixed after 24h cultured microglia on neuronal phagocytosis and 96h fragmentation after injury neuronal growth. Results: the mixed culture of 24h, "Jisuikang" medicated serum in high dose group in the phagocytic index and phagocytic percentage were significantly higher than those in control group (P0.05), middle dose group was lower than that of LPS group (P0.05), the high dose group had no significant difference with LPS group (P0.05). The mixed culture of 96h, "Jisuikang" medicated serum in high dose group level raised more than the number of blank group (P0.05), compared with the LPS group showed no significant difference (P0.05) "Jisuikang" In the high dose group compared with blank group average projection (P0.05), compared with LPS, "Jisuikang" middle dose group the average neurite shorter (P0.05), and high dose group (P0.05) the average long protuberances. Conclusion: the "Jisuikang" through the regulation of microglial phagocytosis promoting repair and regeneration. The damage of neurons, which may be related to apoptosis of material after cleaning, in the microenvironment of the establishment of an invisible protective barrier, but the detailed mechanism still need further research. To sum up, the main innovation of this research lies in: 1 for the first time from the regulation of microglial phagocytosis of SCI was studied in the intervention of traditional Chinese medicine, made a new scientific explanation to.2 research as "Jisuikang" and warming kidney and activating anti SCI effect, creating a variety of methods can accurately, effectively and dynamically observe the microglial phagocytosis of neuronal debris, verify the "Jisuikang" on small Regulation of phagocytosis of glial cells, to a certain extent the effect of nerve regeneration and repair the relationship between the.3 of Chinese medicine intervention "based on the overall concept, thinking and the whole SCI lesions in vitro, the establishment of a multi factor, hybrid system integrity, and conducted a preliminary study.

【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R285.5

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