【摘要】：目的:本研究采用靶向CD147的單克隆抗體對納米基因載體顆粒進行靶向修飾后,進行針對肺癌細胞的蛋白激酶Cε(protein kinase Cε,PKCε)小干擾RNA基因治療,觀察其對肺癌細胞增殖和遷移能力的抑制效果。方法:制作可靶向CD147蛋白的磁性納米基因載體。激光掃描共聚焦顯微鏡觀察肺癌細胞CD147表達量。分別設立CP組、CN組和LP組復合物,按每6孔板孔質�？偭�250 ng進行細胞轉染。另設CD147靶向載體對照CA組和未轉染細胞的對照(control)組。激光掃描共聚焦顯微鏡觀察納米造影劑的細胞內(nèi)吞效果。實時熒光定量PCR檢測PKCε的mRNA表達。Western blot法檢測PKCε、Ki67、MMP3、Wnt1和GAPDH的蛋白表達。平板克隆形成實驗檢測細胞的增殖能力。Transwell法檢測細胞的遷移能力。結果:免疫熒光法染色觀察證實,人肺癌A549細胞的胞膜高表達CD147蛋白。CP組細胞中siRNA高效進入A549細胞,質粒內(nèi)吞效率大于CN組和LP組。CP組、CN組、LP組和CA組的A549細胞中PKCε的mRNA相對表達量分別為control組的(9.76±0.18)%、(98.51±0.32)%、(99.17±0.16)%和(99.68±0.11)%,CP組與control組間的差異有統(tǒng)計學顯著性(P0.05),CN組、LP組與control組間的差異無統(tǒng)計學顯著性。CP組PKCε、Ki-67、MMP3及Wnt1蛋白的表達量明顯降低,CN組和LP組與對照組之間的蛋白表達量的差異無統(tǒng)計學顯著性。CP組的克隆形成數(shù)量明顯少于control組,差異具有統(tǒng)計學顯著性(P0.05)。CN組、LP組和CA組的有效克隆數(shù)量與control組相比差異沒有統(tǒng)計學顯著性。CP組的過膜細胞數(shù)量明顯少于control組,差異具有統(tǒng)計學顯著性(P0.05)。CN組、LP組和CA組的數(shù)量與control組相比差異沒有統(tǒng)計學顯著性。結論:靶向CD147修飾的納米基因載體,可以對肺癌細胞進行高效的PKCε-siRNA基因治療,實現(xiàn)對肺癌細胞增殖和遷移能力的高效抑制。
[Abstract]:Objective: to study the small interfering RNA gene therapy of protein kinase C 蔚 (PKC 蔚) targeting lung cancer cells by targeting the nanoparticles with monoclonal antibody targeting CD147. To observe its inhibitory effect on proliferation and migration of lung cancer cells. Methods: magnetic nanogene vector targeting CD147 protein was prepared. The expression of CD147 in lung cancer cells was observed by laser scanning confocal microscopy. The complexes of CP group CN and LP group were set up, and the cells were transfected according to the total amount of plasmid per 6 hole plate hole of 250 ng. CD147 targeted vector control CA group and untransfected cell control (control) group were used. The endocytosis of nano-contrast agent was observed by laser scanning confocal microscope. The mRNA expression of PKC 蔚 was detected by real-time fluorescence quantitative PCR. Western blot was used to detect the protein expression of PKC 蔚 Ki67 MMP3Cnt1 and GAPDH. The ability of cell proliferation was detected by plate clone formation assay. Transwell method was used to detect cell migration ability. Results: the results of immunofluorescence staining showed that siRNA was highly expressed in A549 cells with high expression of CD147 protein. SiRNA was highly expressed in A549 cells. The relative expression of PKC 蔚 in A549 cells in CN group and LP group. CP group was higher than that in control group (9.76 鹵0.18), (98.51 鹵0.32), (99.17 鹵0.16)% and (99.68 鹵0.11), respectively. There was significant difference between CP group and control group (P0.05). The expression of PKC 蔚 Ki-67MMP3 and Wnt1 protein in CP group was significantly lower than that in control group. There was no significant difference in protein expression between CN group and LP group. There was no significant difference between CP group and control group. The number of clone formation in CP group was significantly lower than that in control group. There was no significant difference in the number of effective clones between LP group and CA group compared with control group (P0.05). The number of overmembrane cells in CP group was significantly lower than that in control group. The difference was statistically significant (P0.05). There was no significant difference in the number of LP group and CA group between CN group and control group. Conclusion: CD147 modified nano-gene vector can effectively inhibit the proliferation and migration of lung cancer cells by PKC 蔚 -siRNA gene therapy.
【作者單位】： 中山大學附屬第三醫(yī)院急診科;中山大學附屬第三醫(yī)院心胸外科;
【基金】：廣東省科技計劃(No.2014A020212533)
【分類號】：R734.2

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