基因芯片筛选人肝癌细胞株MHCC97中血管生成拟态相关基因

Searching for vasculogenic mimicry-associated differentially expressed genes in human hepatocellularcarcinoma cell line MHCC97 using functional grouping cDNA microarray

目的通过观察三维培养条件下高、低转移人肝癌细胞株MHCC97-H、MHCC97-L形成血管生成拟态（VM）的差异，探讨VM形成与细胞外基质和粘连分子相关机制。方法建立MHCC97-H、MHCC97-L三维培养体系，倒置显微镜下观察血管样结构形成差异。以人脐静脉内皮细胞、无转移人肝癌细胞株Hep3B及正常人肝细胞株HL-7702作对照。应用细胞外基质和粘连分子基因芯片筛选MHCC97-H和MHCC97-L中差异表达的基因，并采用RT-PCR和Western blot验证芯片的结果。组间比较采用两样本t检验。结果三维培养24h，MHCC97-H形成的血管样结构长度为（474±16）rmn／cm^2，MHCC97-L为（320±41）mm／cm^2，两者比较，差异有统计学意义（t=6．119，P〈0．05）。Hep3B及HL-7702均不形成血管样结构。在113个细胞外基质和粘连分子相关基因中，MHCC97-H表达较MHCC97-L上调的有7个，下调的有3个。选取差异性表达的腱糖蛋白-C和细胞外基质蛋白1经RT-PCR及Western blot验证，结果与基因芯片结果基本一致。结论高转移人肝癌细胞株MHCC97-H体外形成VM能力明显较低转移人肝癌细胞株MHCC97-L强，其原因可能与MHCC97-H差异表达某些细胞外基质和粘连分子相关基因有关。

Objective To observe vasculogenic mimicry formation difference in human hepatocellular carcinoma cell lines MHCC97-H/L with different metastatic potentials under three-dimensional culture condition, and to study extracellular matrix and adhesion molecules-related mechanism of vasculogenic mimicry. Methods Three-dimensional cell culture system of MHCC97-H/L was established to observe tubelike structures by inverted microscope. Human umbilical vein endothelial cell （HUVEC）, human hepatocellular carcinoma cell line Hep313 with non-metastatic potentials and normal human hepatic cell line HL-7702 were taken as control. Gene expression profiles of MHCC97-H and MHCC97-L were detected by Oligo extracellular matrix and adhesion molecules microarray analysis. Two differentially expressed genes were verified with semi-quantitative reverse transcription- polymerase chain reaction （RT-PCR） and Western blot. All data were analyzed using two sample t test. Results After a 24-hour three-dimensional culture, the length of tubelike structures was （474 ± 16）mm/cm^2 in MHCC97-H, which were significantly longer than （ 320 ＋ 41 ） mm/cm^2 in MHCC97-L （ t = 6. 119, P 〈 0.05 ）. Hep3B anc HL-7702 could not form tubelike structures. Compared with MHCC97-L, the expression of 7 genes were up-regu- lated and the expression of 3 genes were down-regulated among a total of 113 angiogenesis genes in MHCC97-H. Two of the genes with differential expressions including tenascin C and extraeellular matrix protein 1 were further validated by RT-PCR and Western blot. Conclusion MHCC97-H has stronger ability to form vasculogenic mimicry than MHCC97-L, and this perhaps correlates with the differential expression of certain extracellular matrix and adhesion molecules-related genes in MHCC97-H.