| PERFACEBlood - brain barrier (BBB) is the largest barrier for medicines entering brain tissues. Blood - brain tumor barrier (BTB) is between brain tumors and vessels, which limits the delivery of therapeutic drugs to brain tumors. How to open BTB is the key to promote therapeutic drugs into brain tumors and raise therapeutic effects. Recent studies have demonstrated in a rat brain tumor model drug delivery to brain tumor is enhanced following intracarotid infusion of Brady-kinin (BK) , its implement is probably by increasing the qualities of pinocytosis vesicles in the capillary endothelium cells. But concrete mechanism is not clear. Preliminary studies demonstrate calcium - activated potassium channel (KCa) is a important factor for influencing the opening of BTB. The experiment investigates the mechanisms of BTB opening by setting up rat glioma model, infusing BK, KCa channel excitomotor NS1619 , blocking agent IBTX through internal carotid, observing the changes of capillary endothelium under electron microscope, and determining the changes of distribution and content of KCa channel protein in tumor following the infusing of BK.MATERIALS AND METHODS1. Cells and animals Freezed brain glioma cells C6 ( The Department of Neurobiology, China Medical University) , Wistar female rats ( The Center of Experimental Animal, China Medical University) ,their weights are between 180 and 220 gram.2. Agents and instruments 10 % Fetal calf serum, RPMI - 1640 Nutrientmedium, Bradykinin, K^ channel excitomotor NS1619 , blocking agent IBTX (Zhong San Bioengineering Limited Company) , KCa channel antibody ( Sigma Chemical Company) , SABC immunohistochemistry kit (Wu Han Boster Bioengineering Limited Company), SIGM A3 Kl 8 centrifuger (USA), SANYO MCO -15 ACCO2 incubation box (Japan) ,NARISHIGE stereotaxic apparatus (Japan) , LKB electrophoresis apparatus ( Sweden ) , LEICA freezing microtome ( germa-ny).3. Experimental method3.1. Cultivation of rat C6 brain glioma cells The cells are cultured in the nutrient medium RPMI -1640 containing 10 % fetal bovine serum, temperature is 37^ , moisture degree is 100 % ,gas is 5 % CO2.3.2. Establishment of the model of rat C6 brain glioma C6 cells whose concentration are 10 /lOjxl are infused into right rat caudate nucleus by stereotaxic apparatus, its location is 1 mm in front of anterior fontanel, 3 mm beside sagittal suture, 4.5 mm deep.3.3. Manufacture and observation of electron microscope samples After two weeks of tumor transplantation, rats are anesthetized, BK( 10|xg/kg/min) , NS1619(26. 66u,g/kg/min)and mixture of IBTX(2(xg/kg/min)and BK are respectively infused through internal carotid artery, after 10 minute, the rats are killed and brain tumor samples are taken out to make into electron microscopic section, then the changes of pinocytosis vesicles and gap junction in tumor capillary endothelium cells are observed under electron microscope..' 3.4. Western blot After two weeks of tumor transplantation, rats are anesthetized, then rat heads are cut down and tumor samples are taken out after BK( 10(xg/kg)is infused through internal carotid artery. Rats are divided into 6 groups and one group includes 8 rats: A, group of normal rat brain: the normal rat brain of being infused BK for 10 min. B, tumor group of not being infused: transplanted brain glioma rat of not being infused BK;C, D, E, F are respectively the transplanted brain glioma group of being infused BK for 5,10,15,30 min. KCi channel proteins of samples are detected by the method of Western blot, IDV of protein band is measured by the software Chemi Imager 5500 V2.03 and Fluor Chen 2.0.3.5. Immunohistochemistry SABC stain Above samples are taken out and quickly freezed and sliced, KCa channel protein is stained by the method of immunohistochemistry SABC, its positive expression and distribution are observed by microscope, the average optical value of vessel areas is measured by software Motic Image Advanced 3.2.4. Statistical treatment of results One way anova of software SPSS10.0 is used for statistical analysis, average value ± standard deviation is used to indicate data,P<0.05 is statistically significant.RESULTS1. Eectron microscope The increase of pinocytosis bullule is not observed in the capillary endothelium cell of rat brain glioma of not being infused BK and being infused BK + IBTX;The increase of pinocytosis vesicles is observed in the capillary endothelium cell of rat brain glioma of being infused BK and being infused NS1619;Marked change of gap junction is not observed in the capillary endothelium cell of all samples.2. Western blot The band of protein whose molecular mass is 125 KD is obtained from protein electrophoresis, the width and density of every bands are different, the protein band of group of being infused for 10 min is the widest and densest. IDV of groups is expressed with table 1, One way anova is used for statistical treatment, group B is contrast control, group A and F are respectively compared with B, P > 0. 05>, group C, D and E are respectively compared with B, P <0.05 ,group D is respectively compared with group C and E, P <0.05.3. Imunohistochemistry Positive position is mostly in cellular membrane, partly in cytoplasm( subunit a is synthesized in cytoplasm and stained). In normal brain tissue slice, the cell membrane and cytoplasm of capillary endothelium and neurone are positively stained, but their colors are light;In untreated brain gliomas slices , capillary endothelium cells are positively stained, but their colors are light too, less glioma cells are positively stained, moreover, the areas where glioma cells are conferted are hardly stained;In brain glioma slices of being infused BK, capillary endothelium cells are distinguished positively stained,moreover, the stain of the tumor of being infused BK for 10 mins is the most distinguished , the stain of glioma cell is less. Value of average optical of vessel areas is expressed with table 2, One way anova is used for statistical treatment, group B is contrast control, group A and F are respectively compared with B, P > 0. 05, group C, D and E are respectively compared with B, P < 0. 05, group D is respectively compared with group C and E, P <0.05.DISCUSSIONBlood brain barrier ( BBB) lies between normal brain tissue and vessel, which has three layers: capillary endothelium cell, basal lamina and horizontal cell foot process. BBBs function is decided by the specificity of brain capillary endothelium cell. In neurogliocytoma, the membrane is partly destroyed, but there is still blood brain tumor barrier, which still largely confines antitumor drug into tumor tissue.Bradykinin ( BK) is a group that consist of kinin material, it may regulate the releasing of active medium after binding with receptor. BK receptor has Bl and B2. Generally, the most function of BK is mediated by B2 receptor, which is verified in the experiment of inbody and outbody. Using double immunohisto-chemistry stain method, Liu yun hui had verified that brain glioma cell heavy expresses B2R, but there isnl B2R in the capillary endothelium cell of normal brain and tumor tissue. Binding when BK increases BTB's permeability and the BBBs permeability of normal brain tissue is not influenced, it is presumed that BK selectively opening BTB be resulted by a serial of reaction after BK binding B2R. By now, the mechanism on BK selectively opening BTB has not been made clear. It is presumed that the integrality of the BTB in glioma tissue be destroyed, BK easily go through the blood capillary and bind with B2R in glioma cell, which lead to cascade reaction within tumor cell and some vasoactive substance be released from tumor cell, then these vasoactive substance have a role in vessel wall and the permeability of vessel be changed. Actived B2R may increase Ca2 + in endochylema, which would activate NO synthetase to produce NO that activate adenyl cyclase to produce hypso - concentration cGMP. It hadbeen verified that NO and cGMP are key material that increase the BTBs permeability. Ningaraj NS study discovered that the amount of pinocytosis vesicles in rat brain tumor capillary endothelium cell after the infusion of ^channel excito-motor (NS1619) through internal carotid artery, but has no effect in normal brain tissue BBB. BK,NS1619,NO and sGC excitomotor will lead to the increasing of K+ into cell, so, we presume that potassium channel have very important role in the course of biochemistry regulation of BTB permeability.In the experiment, we observe that infusing BK and KCachannel excitomotor NS1619 can increase the amount of pinocytosis vesicles of capillary endothelium cell in tumor area through rat internal carotid artery, i. e. the permeability of capillary vessel has increased. On these grounds, it is presume that the roles of BK and NS1619 be identical. The pinocytosis vesicles of capillary endothelium cell in the tumor sample that is infused KCa channel blocker IBTX after BK through rat internal carotid artery does not increase, On these grounds, it is presume that some relation lie between BK and Ka channel, it is possible that BK can increase the expression of KCa channel protein of capillary endothelium cell and increase the forming of pinocytosis vesicles of capillary endothelium cell, more materials are transported, and BBB is opened. In the experiment, we first time prove that infusing BK through internal carotid artery can increase the expression of KCb channel protein of capillary endothelium cell in tumor tissue and confirm our presumption and reveal the expression of KCi channel protein is possibly one of the mechanisms of BK selectively opening BTB. The firstly, We obtain a band of protein whose molecular mass is 125 KD from western blot protein electrophoresis, it may be seen that their dense and width are different. The normal brain tissue is compared with the untreated glioma tissue, their difference is not statistically significant, which indicates the expression of KCll channel protein in the both has no change, indirectly indicates the BBB is not opened. Untreated glioma is used for contrast control, the IDV of the groups of being infused BK for 5, 10, 15min are respectively compared with it, their differences are statistically significant, which indicates that the BK has have a role in it, it leads to the increasing of expression of KCachannel protein. We still find that the IDV of being infused for 10 min is maximum, which is compared with 5 min and 15 mingroups, their differences have distinguished significance, the result indicate that the expression of KCachannel protein is maximum after being infused for 10 min. The 30 min group is compared with contrast control, the difference, is not significant , which offers clinical theoretical basis, the role of BK has disappointed after being infused for 30 min, BTB is closed, at this time the medicine amount into tumor tissue is possibly extremely little, which do not get to the treating goal. Although we find the expression of KCa channel protein is increased, KCa channels lie in kinds of tissue cells, we do not determine the location of increased KCachannel. So, we use the method of immunohistochamistry SABC to determine the expression location and measure the expression intensity of KCa channel protein in capillary endothelium cell, neuron and glioma cell. The positive expression position of the KCa channel protein is mostly in cellular membrane , partly in cytoplasm, because a subunit is synthetized and stained in cytoplasm. In normal brain tissue slice, the cell membrane and cytoplasm of capillary endothelium and neuron are positively stained, but their colors are light;In untreated brain gliomas slices, capillary endothelium cells are positively stained, but their colors are light too, less glioma cells are positively stained, moreover, the areas where glioma cells are conferted are hardly stained;In brain glioma slices of being infused BK, capillary endothelium cells are distinguished positively stained, moreover, the stain of the tumor of being infused BK for 10 mins is the most distinguished, the stain of glioma cell is less. Immuno-histochemistry staining displays a distribution tendency: the increasing of the expression of KCachannel protein is mostly in capillary endothelium cell after being infused BK. Further, Value of average optical of vessel areas is measured and statistically treated. The normal brain tissue is compared with the untreated glioma tissue, their difference is not statistically significant, which indicates that infusing BK can not increase the expression of KCachannel protein in normal brain tissue, i. e, BBB is not affected. Untreated glioma is used for contrast control, the values of average optical of vessel areas of the groups of being infused BK for 5, 10, 15min are respectively compared with it, their differences are statistically significant, which indicates that the expression of KCachannel protein is increased, moreover the 10 min group is compared with the 5 and 15 min groups,their differences are distinguished significant, the values are further less than 0. 01, which indicates that the expression increasing of KCachannel protein after being infused BK has a relation with the time, i. e. in the 10 min from beginning, the expression of KCachannel protein is increased step by step, at the point of 10 min, the expression gets to the maximum, following it, the expression is decreased, the 30 min group is compared with contrast control, the result is not statistically significant, which the role of BK has disappointed.So, clinically, the effect of infusing chemotherapy medicine at being infused BK for 10 min is the best. The study not only theoretically proves the role of KCachannel in BK selective opening BTB, but also offers the experimental basis for clinical medicine application for treating glioma.CONCLUSION1. Pinocytosis vesicle in the capillary endothelium cell of rat brain glioma is increased by infusing BK and NS1619 through internal carotid artery, which may open BTB.2. The expression increase of KCa channel protein in brain tumor tissue is increased by infusing of bradykinin through rat internal carotid artery, the increase changes with infusing time and the increase in 10 min is most.3. The expression increase of K^ channel protein in brain tumor tissue by infusing of bradykinin through rat internal carotid artery is most in the capillary endothelium cell of rat brain glioma -i4. The expression increase of Kq, channel protein of capillary endothelial cell in glioma is probably one of important factors in BK selective opening blood- brain tumor barrier.
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