大鼠C6脑胶质瘤血—瘤屏障功能结构研究与相关蛋白检测
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摘要
目的:研究C6脑胶质瘤诱导的不同区域BBB通透性改变,从微血管超微结构和功能信号蛋白表达等多环节深入探讨BBB/BBTB通透性增高的主要途径及其调节的潜在机制。
方法:取10μl含1×10~6C6胶质瘤细胞接种健康雄性SD大鼠(体重250～300g)右脑尾状核,建立鼠脑胶质瘤模型。于肿瘤中晚期(接种后3w)处死动物,以C6脑胶质瘤肿瘤中心、肿瘤边缘、BAT(同侧_大脑半球距肿瘤边界≤2mm脑组织)、BDT(同侧大脑半球距肿瘤边界>2mm脑皮质)和NBT(对侧大脑半球脑皮质)作为研究热点区域,通过免疫组织化学方法半定量分析内源性血清白蛋白血管外渗,判定BBB破坏程度;采用硝酸镧透射电镜示踪法和HRP透射电镜示踪法,观察脑毛细血管内皮细胞间TJ和胞饮作用的改变;通过ALP酶组织化学方法测定C6脑胶质瘤的MVD;经免疫组织化学方法测定C6脑胶质瘤MMP-9和iNOS的表达,结合Western-blot法测定TJPs分子occludin表达的改变,进而分析MMP-9、iNOS活性、MVD、occludin与微血管通透性改变之间的关系。
结果:(1)SD大鼠右脑尾状核接种C6脑胶质瘤细胞建立的脑胶质瘤
Objective: Increased permeability of blood brain tumor barrier (BBTB) has been observed in gliomas. This study was performed to investigate the change of BBB permeability in different rigeons of middle-late phase C6 glioma and to evaluate the mechanisms from multiple aspects, by which gliomas induce enhanced BBTB permeability, including ultrastructure of microvessle and functional signal proteins.Methods: (1) Male SDrats (weight 250~350g) were chosen as subjects. 10μl solution containing 1×10~6 C_6 gliomaous cells was sterotactically implanted into the right caudate nuclei to establish an experimental model of brain glioma in SD rats. The rats with brain-bearing C6 glioma were sacrificed at the middle-late phase of glioma, and then the samples were chosen from tumor core, tumor periphery region, BAT (tissues within 2 mm of the border of the tumor), BDT (cortical tissues at least 2 mm away from tumor border but within the ipisilateral hemisphere) and NBT (cortical tissues on the opposite hemisphere of the brain) respectively. (2) By using immunohistochemical SP method , the semiquantitative analysis of extravasation of endogenous albumin was performed to analyse the integrity of endothelial barrier. (3)After the rats were infused intravenously (i.v.) with
various-sized tracers lanthanum nitrate and HRP, the ultrastructure of BBTB in gliomas was observed by lanthanum nitrate-tracing and HRP-tracing transmission electronic microscope to demonstrate the anatomical routes of BBTB leakage. (4) By examining the expression of specific TJ proteins occludin with Western-blot analysis in blood vessels of glioma, the possible involvement of interendothelial TJPs was investigated. (5) MVD was assessed by enzymohistochemistry of alkaline phosphatase (ALP) . (6) The expression of MMP-9 and iNOS were detected immunohistochemiscaly in gliomas to examine the associations between their activity, expression of occludin and permeability of microvessels.Results: (1) After implantation with 1 X 106 C6 gliomaous cells into the right caudate nuclei, all the SD rats developed visible solid glioma during middle-late phase.(2) The intensity of Immunoreaction products of albumin varied among different sectors of the C6 tumor. Albumin positive staining located diffusely among the interstitial spaces of C6 glioma. Moderate and intensive positive reaction of albumin was found in interstitial spaces and necrosis region of tumor, respectively. In the normal brain tissues, the albumin staining was negtive. BAT showed weak positive reaction of albumin and the intensity decreased significantly with the increasing distance to tumor edge.(3) HRP-tracing light microscope results:? Endogenic activity of HRP was only found in the necrosis and bleeding regions.? For the non-tumorous tissues, localization of HRP was restricted in the
capillary lumen and a thin, linear staining along the lumen face of endothelial cells was seen.(3) In C6 glioma, HRP reaction products DAB was located both in the lumen and along the outer wall of capillary in a thick diffusion pattern, consistent with the results of electronic microscope. (4) Electronic microscope results:? In samples of nontumorous brain tissues, low permeability were consistently recorded. HRP or lanthanum is excluded from the tight junction. Both of them were not observed leaking out of the capillaries. (D In brain adjacent to tumor (BAT) , permeability and ultrastructure of tight junctions were comparable to normal brain capillaries. Occasionally, the tight junction was completely infiltrated with lanthanum salt granules or HRP and extravasation to the basement membrane was seen in certain sites of the capillaries.(3)The permeability and ultrastructure of tight junctions in both the periphery sector and core of glomas greatly altered. Lanthanum salt granules and HRP penetrated the cellular gaps of endothelia and seeped in the basement and interstitial spaces, esp for the latter, Lanthanum salt granules distributed extensively among the interstitial spaces and sometimes appeared in the cytoplasm of tumor cell. Moreover, there was continuity of La3+/ HRP deposit from capillary lumen through TJ to the basal lamina and then to the interstitial clefts of the surrounding brain.? There was a significant increase in the number of vesicles in the cytoplasm of tumor capillaries endothelial cells as compared with nontumorous brain tissues(P<0.05). However, very few Lanthanum-filled or
HRP-filled vesicles were seen in the cytoplasm of cerebral microvessel endothelial cells both in tumor and normal brain tissues(P>0.05).(5) In the microvessels of glioma, the opened tight junction could be found. But the basement membrane was continued, compact and lineal. There were tumor cells arranging along the basement membrane, which acted as pericytes. No fenestration on the basement membrane was seen. In addition, there were no glioma cells found in the blood vessels.(5) Microvessel density (MVD) was significantly different between C6 glioma tissues and normal brain tissues (P<0.05) .(6)Positive immunohistochemical staining of iNOS and MMP-9 were all localized in the cytoplasm. In contrast with the non-tumor brain tissues, C6 glioma tissues showed overexpression of iNOS and MMP-9. The expression was remarkable at the edge of tumor tissue and undetectable in normal brian tissue. The positive rate of iNOS expression was 100% in C6 glioma tissues, significantly different from that of nontumorous brain tissues (PO.05), so was the positive rate of MMP-9 expression. Both the expression of iNOS and MMP-9 were positively correlated with the MVD of C6 glioma and negatively correlated with the exvasatation of albumin. The stronger MMP-9 and iNOS expressed, the more albumin appeared in the interstitial of C6 gliomas.(7) Western blot analyses showed occludin bands as broad signal in the normal brain tissues, while the tumor tissues narrowed to a more focused band (apparent size approximately 65 kDa). There was no additional band. Occludin band of both tumor tissues and BAT showed a significant
decrease (35.20% and 7.39%, respectively) compared with normal brain tissues (PO.05). BDT showed no significant change in comparison to normal brain tissues.Conclusion: In conclusion, these data suggest C6 gliomas are accompanied by alteration in BBB, including both functional and ultrastructural aspects. Tight junctions (TJ) between the cerebral microvessel endothelial cells are critical for maintaining brain homeostasis and low permeability of blood-brain barrier (BBB). Regulation of endothelial tight junction at the BBB of glioma is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins.(1) It's reliable to establish an experimental model of brain glioma in SD rats by implanting 1 X 106C6 gliomaous cells sterotactically into the right caudate nuclei.(2) In C6 gliomas, large-sized molecule albumin can pass through the disrupted BBTB and extravasate from microvessels to the local peri-vascular area and distribute diffusely in the interstitial spaces, which may result in peritumoral oedema.( 3 ) It's the intercellular tight junctions that completely restrict extravasation of HRP and lanthanum. Alterations in tight junctions is the major ultrastructural characteristic of tumor-induced capillary endothelium. The disruption of BBB tight junction in G6 glioma might provide the main route of transport of lanthanum nitrate and HRP through endothelial wall. La3+/ HRP probably don't pass through the endothelial cells by transcytosis.(4) The BBB integrity in brain adjacent to tumor (BAT) keeps relatively
well, whose permeability is greatly reduced in comprarison to the tumor. Unfortunately, BAT sometimes consists of invading single tumor cells, which limites the response of brain tumors to systemic chemotherapy.(5) The permeability of regional capillary greatly varied among different brain and among different sectors of the dissected tumor, which may be related to alteration of tight junctions in corresponding sectors.(6) The glioma cells show some atypia, however they still remain part of ability to induce the formation of the BBB. As a result, the glioma cells fail to enter the blood stream and form metastasis in distant areas.(7) The increased MMP-9 and iNOS activity may be correlated with the altered vascular permeability and decreased expression of the tight junction protein occludin in glioma.(8) Occludin changes correlates negatively with alterations in vascular permeability. TJ proteins occludin is involved in the regulation of TJ permeability. Loss of the permeability of BBB was accompanied by decreased expression of TJ proteins.(9) Altered tight junctions are the main structural feature of the disturbed BBB in C6 gliomas and in gliomas in general, which contributes to BBB disruption during gliomas in the rat.
方法:取10μl含1×10~6C6胶质瘤细胞接种健康雄性SD大鼠(体重250～300g)右脑尾状核,建立鼠脑胶质瘤模型。于肿瘤中晚期(接种后3w)处死动物,以C6脑胶质瘤肿瘤中心、肿瘤边缘、BAT(同侧_大脑半球距肿瘤边界≤2mm脑组织)、BDT(同侧大脑半球距肿瘤边界>2mm脑皮质)和NBT(对侧大脑半球脑皮质)作为研究热点区域,通过免疫组织化学方法半定量分析内源性血清白蛋白血管外渗,判定BBB破坏程度;采用硝酸镧透射电镜示踪法和HRP透射电镜示踪法,观察脑毛细血管内皮细胞间TJ和胞饮作用的改变;通过ALP酶组织化学方法测定C6脑胶质瘤的MVD;经免疫组织化学方法测定C6脑胶质瘤MMP-9和iNOS的表达,结合Western-blot法测定TJPs分子occludin表达的改变,进而分析MMP-9、iNOS活性、MVD、occludin与微血管通透性改变之间的关系。
结果:(1)SD大鼠右脑尾状核接种C6脑胶质瘤细胞建立的脑胶质瘤
Objective: Increased permeability of blood brain tumor barrier (BBTB) has been observed in gliomas. This study was performed to investigate the change of BBB permeability in different rigeons of middle-late phase C6 glioma and to evaluate the mechanisms from multiple aspects, by which gliomas induce enhanced BBTB permeability, including ultrastructure of microvessle and functional signal proteins.Methods: (1) Male SDrats (weight 250~350g) were chosen as subjects. 10μl solution containing 1×10~6 C_6 gliomaous cells was sterotactically implanted into the right caudate nuclei to establish an experimental model of brain glioma in SD rats. The rats with brain-bearing C6 glioma were sacrificed at the middle-late phase of glioma, and then the samples were chosen from tumor core, tumor periphery region, BAT (tissues within 2 mm of the border of the tumor), BDT (cortical tissues at least 2 mm away from tumor border but within the ipisilateral hemisphere) and NBT (cortical tissues on the opposite hemisphere of the brain) respectively. (2) By using immunohistochemical SP method , the semiquantitative analysis of extravasation of endogenous albumin was performed to analyse the integrity of endothelial barrier. (3)After the rats were infused intravenously (i.v.) with
various-sized tracers lanthanum nitrate and HRP, the ultrastructure of BBTB in gliomas was observed by lanthanum nitrate-tracing and HRP-tracing transmission electronic microscope to demonstrate the anatomical routes of BBTB leakage. (4) By examining the expression of specific TJ proteins occludin with Western-blot analysis in blood vessels of glioma, the possible involvement of interendothelial TJPs was investigated. (5) MVD was assessed by enzymohistochemistry of alkaline phosphatase (ALP) . (6) The expression of MMP-9 and iNOS were detected immunohistochemiscaly in gliomas to examine the associations between their activity, expression of occludin and permeability of microvessels.Results: (1) After implantation with 1 X 106 C6 gliomaous cells into the right caudate nuclei, all the SD rats developed visible solid glioma during middle-late phase.(2) The intensity of Immunoreaction products of albumin varied among different sectors of the C6 tumor. Albumin positive staining located diffusely among the interstitial spaces of C6 glioma. Moderate and intensive positive reaction of albumin was found in interstitial spaces and necrosis region of tumor, respectively. In the normal brain tissues, the albumin staining was negtive. BAT showed weak positive reaction of albumin and the intensity decreased significantly with the increasing distance to tumor edge.(3) HRP-tracing light microscope results:? Endogenic activity of HRP was only found in the necrosis and bleeding regions.? For the non-tumorous tissues, localization of HRP was restricted in the
capillary lumen and a thin, linear staining along the lumen face of endothelial cells was seen.(3) In C6 glioma, HRP reaction products DAB was located both in the lumen and along the outer wall of capillary in a thick diffusion pattern, consistent with the results of electronic microscope. (4) Electronic microscope results:? In samples of nontumorous brain tissues, low permeability were consistently recorded. HRP or lanthanum is excluded from the tight junction. Both of them were not observed leaking out of the capillaries. (D In brain adjacent to tumor (BAT) , permeability and ultrastructure of tight junctions were comparable to normal brain capillaries. Occasionally, the tight junction was completely infiltrated with lanthanum salt granules or HRP and extravasation to the basement membrane was seen in certain sites of the capillaries.(3)The permeability and ultrastructure of tight junctions in both the periphery sector and core of glomas greatly altered. Lanthanum salt granules and HRP penetrated the cellular gaps of endothelia and seeped in the basement and interstitial spaces, esp for the latter, Lanthanum salt granules distributed extensively among the interstitial spaces and sometimes appeared in the cytoplasm of tumor cell. Moreover, there was continuity of La3+/ HRP deposit from capillary lumen through TJ to the basal lamina and then to the interstitial clefts of the surrounding brain.? There was a significant increase in the number of vesicles in the cytoplasm of tumor capillaries endothelial cells as compared with nontumorous brain tissues(P<0.05). However, very few Lanthanum-filled or
HRP-filled vesicles were seen in the cytoplasm of cerebral microvessel endothelial cells both in tumor and normal brain tissues(P>0.05).(5) In the microvessels of glioma, the opened tight junction could be found. But the basement membrane was continued, compact and lineal. There were tumor cells arranging along the basement membrane, which acted as pericytes. No fenestration on the basement membrane was seen. In addition, there were no glioma cells found in the blood vessels.(5) Microvessel density (MVD) was significantly different between C6 glioma tissues and normal brain tissues (P<0.05) .(6)Positive immunohistochemical staining of iNOS and MMP-9 were all localized in the cytoplasm. In contrast with the non-tumor brain tissues, C6 glioma tissues showed overexpression of iNOS and MMP-9. The expression was remarkable at the edge of tumor tissue and undetectable in normal brian tissue. The positive rate of iNOS expression was 100% in C6 glioma tissues, significantly different from that of nontumorous brain tissues (PO.05), so was the positive rate of MMP-9 expression. Both the expression of iNOS and MMP-9 were positively correlated with the MVD of C6 glioma and negatively correlated with the exvasatation of albumin. The stronger MMP-9 and iNOS expressed, the more albumin appeared in the interstitial of C6 gliomas.(7) Western blot analyses showed occludin bands as broad signal in the normal brain tissues, while the tumor tissues narrowed to a more focused band (apparent size approximately 65 kDa). There was no additional band. Occludin band of both tumor tissues and BAT showed a significant
decrease (35.20% and 7.39%, respectively) compared with normal brain tissues (PO.05). BDT showed no significant change in comparison to normal brain tissues.Conclusion: In conclusion, these data suggest C6 gliomas are accompanied by alteration in BBB, including both functional and ultrastructural aspects. Tight junctions (TJ) between the cerebral microvessel endothelial cells are critical for maintaining brain homeostasis and low permeability of blood-brain barrier (BBB). Regulation of endothelial tight junction at the BBB of glioma is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins.(1) It's reliable to establish an experimental model of brain glioma in SD rats by implanting 1 X 106C6 gliomaous cells sterotactically into the right caudate nuclei.(2) In C6 gliomas, large-sized molecule albumin can pass through the disrupted BBTB and extravasate from microvessels to the local peri-vascular area and distribute diffusely in the interstitial spaces, which may result in peritumoral oedema.( 3 ) It's the intercellular tight junctions that completely restrict extravasation of HRP and lanthanum. Alterations in tight junctions is the major ultrastructural characteristic of tumor-induced capillary endothelium. The disruption of BBB tight junction in G6 glioma might provide the main route of transport of lanthanum nitrate and HRP through endothelial wall. La3+/ HRP probably don't pass through the endothelial cells by transcytosis.(4) The BBB integrity in brain adjacent to tumor (BAT) keeps relatively
well, whose permeability is greatly reduced in comprarison to the tumor. Unfortunately, BAT sometimes consists of invading single tumor cells, which limites the response of brain tumors to systemic chemotherapy.(5) The permeability of regional capillary greatly varied among different brain and among different sectors of the dissected tumor, which may be related to alteration of tight junctions in corresponding sectors.(6) The glioma cells show some atypia, however they still remain part of ability to induce the formation of the BBB. As a result, the glioma cells fail to enter the blood stream and form metastasis in distant areas.(7) The increased MMP-9 and iNOS activity may be correlated with the altered vascular permeability and decreased expression of the tight junction protein occludin in glioma.(8) Occludin changes correlates negatively with alterations in vascular permeability. TJ proteins occludin is involved in the regulation of TJ permeability. Loss of the permeability of BBB was accompanied by decreased expression of TJ proteins.(9) Altered tight junctions are the main structural feature of the disturbed BBB in C6 gliomas and in gliomas in general, which contributes to BBB disruption during gliomas in the rat.
引文
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2. Gunnersen JM, Spirkoska V, Smith PE, et al. Growth and migration markers of rat C6 glioma cells identified by serial analysis of gene expression. Glia. 2000 Nov;32(2):146-54.
3.吴国祥,余绍祖,李涛,等.大鼠脑胶质瘤的形态学观察,卒中与神经疾病杂志2001第8卷第3期:159～160.
4.余永强,陈骏,钱银锋,等.立体定向建立大鼠C6脑胶质瘤模型,中国药理学报,2003,19(2):222～5.
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