The Study Of Expressions Of Aquaporins And Kir 4.1 In Astrocytic Tumors

PART ONE EXPRESSIONS OF AQUAPORINS AND KIR 4.1 IN HUMAN ASTROCYTIC TUMORSObjectiveThe aim of the present study was to study the expressions of Aquaporins(AQPs) and Inwardly rectifying potassium channels 4.1(Kir 4.1) in human astrocytic tumors and control group, so as to explore the molecular mechanism of astrocytic tumors proliferation and growth.Methodsâ‘ One hundred patients with primary brain astrocytic tumors were included in this study. All these cases were surgically treated at the Neurosurgical Department of the first Hospital affiliated to the Chongqing Medical University, the Neurosurgical Department of the second Hospital affiliated to the Chongqing Medical University, the Neurosurgical Department of Southwest Hospital of the third military medical university and the Neurosurgical Department of Chongqing Emergency Medical Center. The brain tissues in decompression procedure after trauma were served as control group.â‘¡All the patients were detected by Magnetic Resonance Imaging (MRI) before operation.â‘¢One part of tumor specimens was applied for histopathologic diagnosis, TUNEL analysis and Immunohistochemistry, the other part of specimens was applied for Western blot and RT-PCR analysis.â‘£The immunohistochemical method was used to detect the expressions of AQPs and Kir 4.1 in astrocytic tumors and control group.⑤The Western blot analysis was used to detect the expressions of AQPs and Kir 4.1 protein in astrocytic tumors and control group.â‘¥The RT-PCR analysis was used to detect the expressions of AQPs and Kir 4.1 mRNA in astrocytic tumors and control group.⑦All statistics were performed using the SAS 9.1 software package (Chongqing Medical University, China).Resultsâ‘ Most human astrocytic tumors were located in frontal lobe and temporal lobe by MRI detecting.â‘¡Pathologic grades and histologic subtypes were defined according to the 2000 WHO criteria as follows: 25 cases with grade IV, 27 cases with grade III, 29 cases with grade II, 19 cases with grade I.â‘¢The apoptosis index was decreased with the ascending pathologic grade( P<0.05 ).â‘£Compared with the control group, the expression of Kir 4.1 in astrocytic tumors was stronger(p < 0.05). With the ascending pathologic grade, the expression of Kir 4.1 in tumor specimens significantly increased (p < 0.05).⑤Compared with the control group, the expression of AQP9 in astrocytic tumors was stronger(p < 0.05). With the ascending pathologic grade, the expression of AQP9 in tumor specimens significantly increased (p < 0.05).â‘¥Compared with the control group, the expression of AQP4 in WHOâ…¢^â…£astrocytic tumors was stronger(p < 0.05), the expression of AQP4 in WHOâ… ^â…¡astrocytic tumors was weaker(p < 0.05). The expression of AQP4 in WHOâ…¢^â…£astrocytic tumors was stronger than that in WHOâ… ^â…¡tumors(p < 0.05).⑦Compared with the control group, the expressions of AQP3 and AQP5 in astrocytic tumors were stronger(p < 0.05). With the ascending pathologic grade, the expressions of AQP3 and AQP5 in tumor specimens were not changed(P>0.05).⑧Compared with the control group, the expression of AQP8 in astrocytic tumors was not different(P>0.05).Conclusionsâ‘ The expression of Kir 4.1 in astrocytic tumors was correlated with pathologic grade. Kir 4.1 may play an important role in malignant progression of brain astrocytic tumors, acting as a useful biomarker for the molecular diagnosis and a new candidate target for gene therapy of astrocytic tumors.â‘¡The expression of AQP9 in astrocytic tumors was correlated with pathologic grade. AQP9 may play an important role in malignant progression of brain astrocytic tumors, acting as a useful biomarker for the molecular diagnosis and a new candidate target for gene therapy of astrocytic tumors.â‘¢AQP4 may not play an important role in water transportation of WHOâ… ^â…¡brain astrocytic tumors.â‘£AQP3 and AQP5 may play important roles in water transportation of brain astrocytic tumors, The underlying mechanism that why expressions of AQP3 and AQP5 among astrocytic tumors were not correlated with pathologic grade remains to be further clarified.⑤The molecular mechanisms that whether AQP8 affects the proliferation and apoptosis of astrocytic tumors are to be further studied. PART TWO EXPRESSIONS OF AQUAPORINS AND KIR 4.1 IN THE CELL CYCLE OF RAT GLIOMA CELL LINE C6ObjectiveThe aim of the present study was to study the expressions of AQPs and Kir 4.1 in the cell cycle of rat glioma cell line C6, so as to explore the molecular mechanism of glioma proliferation and growth. Methodsâ‘ Rat glioma cell line C6 was undergone the culture, astrocytes undergone the primary culture were served as control.â‘¡The cells were divided into G₀/G₁ phase, S phase and G₂/M phase by synchronization. The effects of synchronization were detected by flow cytometry.â‘¢Transmission electron microscopy(TEM) was used to detect the ultrastructure of C6 cells.â‘£The immunofluorescence staining method was used to detect the expressions of AQPs and Kir 4.1 in cells in different phases, and the co-expressions of AQP4 and Kir 4.1 was detected by double immunofluorescence staining.⑤The Western blot analysis was used to detect the expressions of AQPs and Kir 4.1 in cells in different phases. â‘¥All statistics were performed using the SAS 9.1 software package (Chongqing Medical University, China).Resultsâ‘ The fraction of C6 glioma cells was increased to 80.20%, 65.36%, 92.47% respectively in S phase, G₂/M phase and G₀/G₁ phase, the fraction of astrocytes was increased to 78.57%, 62.10%, 90.37% respectively in S phase, G₂/M phase and G₀/G₁ phase after synchronization.â‘¡The shape of C6 cells was irregularity. The endoplasmic reticulum accrementition, cytolymph abundance, chromatin margination, swollen mitochondrion and nucleolus irregularity were detected by TEM.â‘¢The expressions of AQP3, 5, 8, 9 in G₀/G₁ phase were strongest than those in S phase and G₂/M phase in C6 cells and astrocytes(P<0.05). There was no difference between The expressions of AQP3, 5, 8, 9 in S phase and those in G₂/M phase(P>0.05). The expressions of AQP3, 5, 9 in C6 cells were stronger than those in astrocytes in different phases(P<0.05). There was no difference that the expression of AQP8 in different phases in C6 cells and astrocytes(P>0.05).â‘£The expression of AQP4 was detected in G₀/G₁ phase, but there was no expression in S phase and G₂/M phase in C6 cells and astrocytes. The expression of AQP4 in C6 cells was stronger than that in astrocytes in G₀/G₁ phase(P<0.05).⑤The expression of Kir 4.1 in G₀/G₁ phase was strongest than that in S phase and G₂/M phase, and the expression of Kir 4.1 in G₂/M phase was stronger than that in S phase in C6 cells and astrocytes(P<0.05). The expression of Kir 4.1 in C6 cells was stronger than that in astrocytes in G₀/G₁ phase, the expression of Kir 4.1 in astrocytes was stronger than that in C6 cells in G₂/M phase (P<0.05). There was no difference that the expression of Kir 4.1 in S phase in C6 cells and astrocytes(P>0.05).â‘¥The co-expressions of AQP4 and Kir 4.1 in C6 cells were stronger than those in astrocytes in G₀/G₁ phase (P<0.05). The ratio of Kir 4.1/AQP4 in C6 cells was larger than that in astrocytes(P<0.05).Conclusionsâ‘ AQP3, 5, 8, 9 may play important roles in G₀/G₁ phase in C6 cells and astrocytes, AQP3, 5, 9 may play important roles in water transportation of C6 glioma cell line, but AQP8 may not play important roles in water transportation of C6 glioma cell line.â‘¡The expression of AQP4 is dependent upon the cell cycle, it only expressed in G₀/G₁ phase in C6 cells and astrocytes. The expression of AQP4 in C6 cells was stronger than that in astrocytes, AQP4 may play an important role in water transportation of C6 glioma cell line.â‘¢Kir 4.1 may play an important role in G₀/G₁ phase in C6 cells, Kir4.1 expression might shift a significant number of cells from the G₂/M phase into the G₀/G₁ stage of the cell cycle, but the mechanism remains to be further clarified. â‘£The co-expressions of Kir 4.1 and AQP4 in C6 cells was stronger than those in astrocytes, but the expression of Kir 4.1 was much stronger. The function of Kir 4.1 and AQP4 might be uncoupled, but the mechanism needs to be further studied.PART THREE EXPRESSIONS OF AQUAPORINS AND KIR 4.1 IN THE C6 CELL LINE BRAIN GLIOMA MODELS IN RATSObjectiveThe aim of the present study was to study the expressions of AQPs and Kir 4.1 in the C6 cell line brain glioma models in rats, so as to explore the molecular mechanism of glioma proliferation and growth. Methodsâ‘ 120 Wistar rats were randomly divided into 4 groups (A, B, C, D groups). The tumor groups were A group(1 week after establishing models), B group(2 weeks after establishing models), C group(3 weeks after establishing models). D group was served as control.â‘¡The C6 cell line brain glioma models in rats were established after the C6 cells was stereotaxically injected into the right caudate guided by stereotactic frame. The moving and survival states of rats with glioma were observed.â‘¢All the rats of different groups were detected by Magnetic Resonance Imaging (MRI).â‘£The water content of the brain tissues at the diffenert groups was detected.⑤The characteristic of rat brain C6 glioma was detected by HE staining and immunohistochemical staining of GFAP.â‘¥The immunohistochemical method was used to detect the expressions of AQPs and Kir 4.1 in different groups.⑦The Western blot analysis was used to detect the expressions of AQPs and Kir 4.1 protein in different groups.⑧The RT-PCR analysis was used to detect the expressions of AQPs and Kir 4.1 mRNA in different groups.⑨All statistics were performed using the SAS 9.1 software package (Chongqing Medical University, China).Resultsâ‘ The symptom of the rats was emerged to be at 2 weeks after stereotactic implantation, and were aggravated during 2 to 3 weeks after stereotactic implantation.â‘¡79 rats with tumor growth were clarified by MRI and pathological diagnosis. The average diameter of tumor and the volume of tumor were increased with tumor growth in A, B, C groups by MRI detecting(p < 0.05).â‘¢The water content of the brain tissues was increased with the tumor proliferation(p < 0.01).â‘£Compared with the control group(D group), the expressions of Kir 4.1, AQP3, 4, 5, 9 protein and mRNA in tumor groups(A, B, C groups) were stronger(p < 0.05). There was no difference between the expressions of AQP8 protein and mRNA in tumor groups and those in control group(P>0.05).⑤The expressions of Kir 4.1, AQP4, 9 protein and mRNA in A group was strongest than those in B and C groups, the expressions of Kir 4.1, AQP4, 9 protein and mRNA in B group was stronger than those in C group(p < 0.05). There was no difference that the expressions of AQP3, 5, 8 protein and mRNA in A, B, C groups (P>0.05).Conclusionsâ‘ The rat brain C6 glioma could analogue the human brain tumor because of its invasive character and achievement ratio.â‘¡Kir 4.1, AQP3, 4, 5, 9 might play important roles in water transportation of C6 glioma growth and proliferation.â‘¢The alterations of the expressions of Kir 4.1, AQP4 and AQP9 might be one of the molecular mechanism of C6 glioma proliferation and growth.â‘£The molecular mechanisms that whether AQP8 affects the water transportation in C6 glioma and the proliferation and growth of C6 glioma are to be further studied.