LIF基因转染的ES细胞生长与分化特性的研究

我们将人D型LIF cDNA以正反两种方向分别克隆到载体pKCR 3,并引入neo~r基因,构建成pSVLD( )和pSVLD(-)质粒,按磷酸钙沉淀法分别转染ES-5胚胎干细胞,经G418和不同浓度LIF条件培液共同筛选、Nor-thern和Southern分析以及ES-5细胞集落分化抑制能力测定,建立了过度表达分泌LIF的ESL( )细胞株和表达外源反义LIF RNA的ESL(-)细胞株。我们发现,ESL( )A2细胞能够在无外源LIF常规培液下至少传13代以上,仍能正常生长和传代,并保持与ES-5细胞同样的体外生长的特征性形态,以及具有干细胞特点和发育多潜能性,表明过度表达LIF确实能使ES细胞完全脱离对外源LIF条件培液的依赖性;而表达反义LIP RNA的ESL(-)细胞对培液中LIF浓度的依赖性明显升高,也更易分化,说明ES细胞内源LIF基因的表达水平虽低,但对于抑制ES细胞的分化仍可能是必需的。形态学观察发现,体外悬滴培养中经10~(-6)mol/L RA诱导后,过度表达LIF并未产生抑制ESL( )A2细胞分化的现象,和亲本ES-5细胞比较,也未发现其明显改变了10~(-6)mol/L RA对ESL( )A2细胞诱导分化的方向;而相同条件下,表达外源反义LIF RNA,则使ESL(-)B5细胞更易于向形态明确的细胞包括成纤维样和梭样细胞分化。上述细胞株的建立,提供了一个研究在不添加LIF的常规培液中生长的ES细胞或表达外源反义LIF RNA的ES细胞的生长分化的模型。

STUDY ON GROWTH AND DIFFERENTIATION OF ES CELLS TRANSFECTED WITH LIF GENE

We constructed plasmids pSVLD ( ) and pSVLD( - ) containing human D-form Leukemia Inhibitory Factor (LIF) cDNA sequence in sense or antisense orientation, transfected them into cells of an embryonic stem cell line ES-5, and isolated 248 pSVLD( )-transfected and 93 pSVLD( - )-transfected G 418-resistant clones. By stepwise reducing LIF concentration in the medium, we obtained 3 pSVLD ( )-transfected clones (A 1-3) that could grow in 15% BRL-CM, including ESL( )A2 that could grow without LIF; we also obtained 13 pSVLD(-)-transfected clones (B 1-13) which would differentiate in 60% BRL-CM, including ESL( - )B 3 and B 5 that could not be passaged without LIF. ESL ( ) A 2 and ESL( - ) B 5 cells had the relatively stronger LIF mRNA or antisense LIF RNA expression, and LIF overexpression in ESL( )A 2 cells was shown by biological assay for ES cell differentiation inhibition. ESL( )A 2 cells could be continuously passaged for at least 13 passages without addition of exogenous LIF, retained undifferentiated morphology as well as a high growth rate, and resembled ES-5 cells in terms of stem cell characteristics and pluripotent properties, as analyzed for alkaline phosphatase activity and with staining the paraffin sections of tumor formed by inoculating ESL( )A 2 cells into mouse. On the contrary, ESL( - ) cells should be cultured in higher concentration of LIF than ES-5 cells, otherwise, would undertake extensive differentiation. By hanging drop culture for 3 days in the presence of 10-6mol/L RA then observing the differentiation of the formed embryonic bodies(EBs), we found that ESL( ) A2 and ES-5 cells underwent similar morphologically differentiation, with round and epitheliallike cells occurring around the EBs; while ESL (-) B 5 cells, despite initial differentiation to round cells, differentiate into fibro-blast-like and spindle shaped cells. The above results indicate that LIF overexpression in ESL( )A 2 cells is able to completely free ES cells from the dependence on LIF-conditioned medium, and endogenous LIF gene expression, although is very low, may be indispensable for inhibiting the differentiation in vitro of ES cells; LIF overexpression might not obviously change the differentiation way of ES-5 cells, however, blocking endogenous LIF expression gives rise to the increased sensitivity of ES-5 cells to differentiate, with an altered differentiation pattern. The establishment of ESL( ) and ESL (-) cell lines provides models for further study of the growth and differentiation of ES-5 cells.