Zhang D L, Huang G S, Li L J, He X Y, Xia G T, Gao B X, Bai X H, Liu S G
Peking University Health Science Center, Beijing 100083, China.
Yi Chuan Xue Bao. 2000;27(12):1057-71.
Under the prerequisite that the incidence of cancer or tumor in negatively-controlled nude mice inoculated subcutaneously with feline or canine kidney cell cultures purified in vitro at passage 3 or higher (the modal chromosome number of FKC on passage 3 was 38 of diploid at the rate of 80%) was 0%(0/22) and 0%(0/10) respectively, and the incidence of progressively negative growing tumor in controlled nude mice inoculated subcutaneously with repeatedly-frozen- and thawed-HeLa cell cultures of X strain was 20%(1/5), the negative growing malignant tumor (MT) was found in half of the nude mice inoculated subcutaneously with HeLa cell cultures of H strain(with modal chromosome number of 78 +/- 2 of sub-tetraploid at the rate of 40%), the progressively-growing malignant tumor was found in all the other 40 nude mice inoculated subcutaneously with HeLa cell cultures of other strains, with the incidence of MT in nude mice with KB strain (with modal chromosome number of 60 +/- 3 of hyperdiploid at the rate of 72%-76%) 10/10, the incidence of poorly-differentiated MT originated from epithelia in nude mice with X strain (with modal chromosomal number of 62 +/- 3 of hyperdiploid at the rate of 69%) 25/25, and the incidence of MRT in nude mice with in vitro cultured tumor cell NM20/X strain (with modal chromosome number of 68 +/- 3 of both hyperdiploid and subtetraploid at the rate of 52%) 5/5. After being continuously cultivated for 20 passages in vitro, HeLa cell of X strain was subcutaneously inoculated into nude mice and cultivated for 1 passage in vivo within 15 days, and then the developed growing MT was collected as HeLa cell of NM20/X strain on passage 0 and continuously cultivated for 11 passages to prepare for transplanting into nude mice again. Therefore, the highly variable strain of HeLa cells can be successfully selected by alternate cultivation in vitro and in vivo. Occasionally in another experiment, the progressively-growing MRT was found in all the 4 nude mice of one test group inoculated subcutaneously with 0.17 ml cell-cultures of super-high density containing 12.75 x 10(7) HeLa cells of KB strain on passages 10-11(with the rate of chromosome aberration high to 20% on passages 10-11 including 18% dicentric chromosome and 2% breakage chromosome). Although the incidence of MRT in nude mice inoculated subcutaneously with violently variable HeLa cells of NM20/X strain on passage 11, HeLa cells of KB strain on passages 10-11 reaches 100%(5/5) & 100%(4/4) respectively, yet it is requested that the inoculated live cell number is huge (5-12 x 10(7) cells per nude mouse), the tumor emerges immediately, develops violently, grows very fast, and has an extremely aggressive malignancy, the tumor is rich in the blood vessel giving a full supply of blood for it, and the mean value of major diameter X minor diameter of the tumor is essentially up to the standard of 30 mm x 20 mm in 16-22 days after the inoculation of the cells into the nude mice. The first finding of MRT in model animals provides an opportunity for answering the origin problem of MRT. Based on this reason, human uterus vertical epithelium may be an original tissue of MRT, thus opening up a new era for the research of MRT origin. It is also concluded as follows: 1. Cellular tumorigenicity is different among differently-karyotypic cells. 2. Highly variable strain of tumor cell line can be selected quickly and successfully in nude mouse. 3. Cellular tumorigenicity may be increased if chromosome aberration is very high. 4. The genetic characteristics of chromosomes of HeLa cells determines their tumorigenicity, chromosome number variation of HeLa cells has positive relationship with their carcinogenesis or tumorigenicity, and the turn of HeLa cells concerning their tumorigenicity from weak to strong is KB, X and NM20/X strains (excluding H strain, in which tumorigenicity remains to be determined by further experiments) respectively.
将体外传代3次或更高代次纯化的猫或犬肾细胞培养物皮下接种到阴性对照裸鼠中,癌症或肿瘤的发生率分别为0%(0/22)和0%(0/10)(第3代FKC的模式染色体数为二倍体38条,比例为80%);将反复冻融的X株HeLa细胞培养物皮下接种到对照裸鼠中,进行性阴性生长肿瘤的发生率为20%(1/5);在皮下接种H株HeLa细胞培养物(模式染色体数为亚四倍体78±2条,比例为40%)的裸鼠中,一半出现阴性生长恶性肿瘤(MT);在皮下接种其他株系HeLa细胞培养物的另外40只裸鼠中均出现进行性生长恶性肿瘤,KB株(模式染色体数为超二倍体60±3条,比例为72%-76%)裸鼠中MT的发生率为10/10,X株(模式染色体数为超二倍体62±3条,比例为69%)裸鼠中起源于上皮的低分化MT的发生率为25/25,体外培养的肿瘤细胞NM20/X株(模式染色体数为超二倍体和亚四倍体68±3条,比例为52%)裸鼠中MRT的发生率为5/5。X株HeLa细胞在体外连续传代20次后,皮下接种到裸鼠中并在15天内在体内传代1次,然后将长出的生长型MT作为第0代NM20/X株HeLa细胞收集,并连续传代11次,准备再次移植到裸鼠中。因此,通过体外和体内交替培养可以成功选择出HeLa细胞的高变异性株系。偶尔在另一项实验中,在一个试验组的所有4只裸鼠中均发现进行性生长的MRT,这些裸鼠皮下接种了0.17 ml超高密度细胞培养物,其中含有第10-11代KB株HeLa细胞12.75×10⁷个(第10-11代染色体畸变率高达20%,包括18%的双着丝粒染色体和2%的断裂染色体)。尽管皮下接种第11代NM20/X株剧烈变异HeLa细胞、第10-11代KB株HeLa细胞的裸鼠中MRT的发生率分别达到100%(5/5)和100%(4/4),但要求接种的活细胞数量巨大(每只裸鼠5-12×10⁷个细胞),肿瘤立即出现,发展剧烈,生长非常快,具有极强的侵袭性恶性,肿瘤血管丰富,为其提供充足血液供应,在将细胞接种到裸鼠中16-22天后,肿瘤的长径×短径平均值基本达到标准(30 mm×20 mm)。在模型动物中首次发现MRT为回答MRT的起源问题提供了契机。基于此原因,人子宫垂直上皮可能是MRT的原始组织,从而为MRT起源的研究开辟了一个新时代。还得出以下结论:1. 不同核型的细胞其细胞致瘤性不同。2. 在裸鼠中可以快速成功地选择肿瘤细胞系的高变异性株系。3. 如果染色体畸变率非常高,细胞致瘤性可能会增加。4. HeLa细胞的染色体遗传特征决定其致瘤性,HeLa细胞的染色体数变异与其致癌性或致瘤性呈正相关,HeLa细胞致瘤性从弱到强的顺序分别为KB、X和NM20/X株系(不包括H株系,其致瘤性有待进一步实验确定)。