同一组织来源的肝癌干细胞分化能力异质性的体外研究

目的通过比较肝癌干细胞的不同单细胞克隆的体外分化能力,分析同一组织来源的肿瘤干细胞分化能力是否具有异质性,以进一步明确肿瘤干细胞的分化特性,为靶向肿瘤干细胞的治疗提供实验依据。方法通过有限稀释法对肝癌干细胞进行单细胞克隆培养,获得的单细胞克隆通过MTT测定比较其增殖能力。然后分别挑选增殖速度较快和较慢的3个克隆,采用RT-PCR方法比较其干细胞标志物表达水平。对干细胞标志表达水平高的3个克隆(A2、A3和B2)进行向成骨、软骨和脂肪方向的诱导分化。采用实时荧光定量PCR方法检测诱导前后成骨、软骨和脂肪标志分子的表达。结果获得的20个单细胞克隆增殖能力不同,并且增殖能力快的克隆表达干细胞标志物CD133、Oct4、c-kit、SCF、nestin比增殖能力慢的克隆强。向成骨方向诱导后,A2、A3和B2克隆I型胶原mRNA表达水平分别升高了(4.71±0.11)、(2.13±0.15)和(3.82±0.3)倍;骨钙素mRNA的表达水平分别升高(8.55±0.18)、(7.02±0.03)和(7.91±0.09)倍,说明A2克隆向成骨细胞分化能力最强。向软骨方向诱导后,B2分化能力最强,软骨标志分子蛋白聚糖和II型胶原的表达分别上调(25.01±0.19)倍和(17.49±0.19)倍,而在A2未发生明显变化,A3只轻微上调。向脂肪方向诱导后,A2、A3和B2克隆脂联素mRNA表达水平分别升高了(6.12±0.15)、(11.45±0.36)和(12.41±1.03)倍,过氧化物酶体增殖物激活受体γmRNA的表达水平分别升高(4.92±0.02)、(9.54±0.18)和(8.96±0.11)倍。结论来源于同一组织的肝癌干细胞在分化能力上具有异质性,这可能是导致肿瘤组织异质性的原因之一,也提示靶向肿瘤干细胞的治疗需要联合用药。

Differentiation heterogeneity of liver cancer stem cells derived from the same cancer tissue in vitro

Objective To investigate the differentiation heterogeneity of cancer stem cells （CSCs） by comparing differentiation potential of clonally expanded subpopulations derived from the same cancer tissue in vitro. Methods Single cell clones of CSCs were obtained by limited dilution method. The proliferation of these clones was evaluated by MTT assay. The expression of stem cell markers was detected by RT-PCR. Three clones （A2, A3 and B2） were induced to differentiate toward mesenchymal-like cell for 3 weeks. Real-time PCR was used to detect the mRNA levels of mesenchymal-specific markers. Results The proliferation of the obtained 20 clones was differed between each other, and the expression of stem cell markers CD133, Oct4, c-kit, stem cell factor and nestin was higher in highly proliferation of clones than in the low proliferation of ones. Upon induction toward osteoblast, the mRNA level of collagen type I in clone A2, A3 and B2 was up-regulated to （4.71 ± 0.11）,＆amp;nbsp;（2.13 ± 0.15） and （3.82 ± 0.3） times respectively;and the mRNA level of osteocalcin was up-regulated to （8.55 ± 0.18）, （7.02 ± 0.03） and （7.91 ± 0.09） times respectively, which showed that A2 has stronger potential toward osteoblast. Upon induction toward chondrocyte, clone B2 exhibited very strong differentiation potential as shown the mRNA level of aggrecan and collagen type II was up-regulated to （25.01 ± 0.19） and （17.49 ± 0.19） times. However, the expression of aggrecan and collagen type II was not increased in clone A2 and increased slightly in clone A3. Upon induction toward adipocyte, the mRNA level of adiponectin in clone A2, A3 and B2 was up-regulated to （6.12 ± 0.15）, （11.45 ± 0.36） and （12.41 ± 1.03） times respectively;and the mRNA level of peroxisome proliferator-activated receptor （PPARγ） was up-regulated to （4.92 ± 0.02）, （9.54 ± 0.18） and （8.96 ± 0.11） times respectively, which suggested that A3 and B2 had stronger differentiation potential toward adipocyte. Conclusions The CSCs derived from the same cancer tissue has heterogeneity in differentiation potential, which may be one reason of tumor heterogeneity. These results indicate that combination of drugs might be needed in targeting CSCs treatment in the future.