| IntroductionLeukemia is a familiar malignant carcinoma, which often invades into brain at the end. The incidence of metastasis is about 25 to 81 percent. So metastasis to brain is the main lethal cause of leukemia. From nowdays research we could conclude leukemia cells invade into brain by crossing BBB while the mechanism of it is not clear. To illuminate the mechanism is an important step to prevent occurrence of leukemia. HBMECs and tight junctions between them are basic structure of BBB and tight junction is the crucial part of controlling the permeability of BBB. p160ROCK is a serine/threonine kinase and a privotal member of Rho family which could reorganize cell skeleton . As the importance of cell skeleton in the forming process of TJ, we do some research on HL - 60 cells crossing of BBB.Method1. Building BBB model constructed by HBMEC and counting HL - 60 cells transmigrating it at 8h, 16h,24h,32h and 40h. Suggesting HL -60 cells could transmit BBBand invade into brain.2. After the interaction of HL - 60 and HBMEC , Observing changes of cells skeleton ( Rhodamine - conjugated phalloidin) and TJ ( anti - ZO - 1 antibody of mouse, FITC - conjugated anti -mouse IgG)by fluorescence.3. Detecting the effect of Y - 27632 on HBMEC skeleton and TJ and the process of HL - 60 cells transmigrating of BBB model3. 1 Building BBB model constructed by HBMEC treated with p160ROCK special inhibitor - Y - 27632 and counting HL - 60 cells transmigrating it at 8h,16h,24h,32h and 40h.3. 2 Detecting the effect of Y - 27632 on HBMEC skeleton and TJ by fluorescence4. Constructing positive cell line of stable expression of p160R CK dominant negative4. 1 Identifying plasmid pl60ROCK dominant negative cleaved by enzyme Xbal.4.2 Identifying positive cell clones by fluorescence ( anti - myc antibody of mouse ).4. 3 Building BBB model constructed by HBMEC transfected with p160ROCK dominant negative and HBMEC transfected with vector PCAG. Count HL - 60 cells transmigrating the two BBB models.4.4 Observing skeleton and TJ of transfected cells.5. Detecting effect of the other signal pathway on HBMEC skeleton and TJ ( detecting expression level of AKT and P - AKT by Western blot)Result1. HL - 60 cells transmigrating the BBB model made up of HBMEC are higher and higher as time went by at 8h,16h,24h,32h and 40h.2. After the interaction of HL - 60 and HBMEC for 24h, ZO -1 continuous expression was changed to be uncontinuous by fluorescence detecting, which suggested that HL - 60 cells crossed BBB by opening TJ. Stress fiber was zigzag and became thicker than that of normal HBMEC.3. HL - 60 cells transmigrating the BBB model made up of HBMEC treated with p160ROCK inhibitor - Y -27632 are also higher and higher as time went by. And this group was much higher than the group of HBMEC. Suggesting p160ROCK could adjust the permeability of BBB.Dealed with 10uM Y -27632 for 30 minutes , ZO -1 continuous expression was changed to be dotlike, which suggested that pl60ROCK could adjust TJ. There are some F - actin on the edge of HBMECs . F - actin was hardly seen in HBMECs,which suggested that p160ROCK could reorganize HBMEC skeleton and adjust TJ.4. Constructing positive cell line of stable expression of p160ROCK dominant negative4. 1 Identifying plasmid pl60ROCK dominant negative cleaved by enzyme Xbal and two bands of 1291bp and 6821bp were acquired.4. 2 Acquiring positive cell clones of p160ROCK dominant negative by fluorescence.4. 3 HL - 60 cells transmigrating BBB model of HBMEC transfected with p160ROCK dominant negative are also higher than those of HBMEC transfected with vector PCAG, which suggested p160ROCK could decreased the permeability and inhibited HL -60 crossing BBB.4.4 By fluorescence observing that stress fiber of cells transfected with p160ROCK dominant negative became thinner even disappeared and TJ turned to be uncontinuous, which strongly suggested p160ROCK could reorganize HBMEC skeleton and adjust TJ.5. AKT expression...
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