摘要
研究背景
近年关于H／RS细胞的起源、形态发生、及其生长和调节的分子机制研究有了很大的进展，研究表明H／RS细胞的起源绝大部分来自“残疾B淋巴细胞”。潜伏膜蛋白-1(LMP-1)的表达、CD99表达缺失和核因子κB(NF-κB)的持续活化是H／RS细胞形成的三大要素。最引人注目的是新近发现H／RS细胞的发生与mic2／CD99基因表达缺失有关。国外研究报道将反义mic2／CD99基因转染人B淋巴细胞瘤株BJAB和IM9细胞使CD99表达缺失，能显示出像HL一样的H／RS细胞的典型特征，出现H／RS样“镜影细胞”，高表达CD30和CD15；当再将mic2／CD99基因转入上述H／RS样细胞中，又可使出现的H／RS样细胞的形态特征消失，该细胞又恢复到B淋巴细胞瘤原有的表型特征，提示H／RS细胞的发生及免疫表型的变化与mic2／CD99基因表达缺失有关。研究表明，抑制细胞中CD99的表达，可以诱导具有HL组织学特征的H／RS细胞的产生。
人CD99是一种分子量为32kDa的细胞表面跨膜糖蛋白，编码基因mic2位于X和Y染色体短臂假染色体区(PAR)的Xp22.32-pter和Yp11-pter上，连锁分析提示PAR是HL的潜在致瘤基因，参与血细胞的分化等。本课题组前期研究将小鼠B淋巴瘤细胞株mCD99L2基因沉默，下调CD99的表达，可以诱导H／RS样细胞的产生，并从免疫表型、生物学特性等方面验证其H／RS细胞部分特性。如果上调人HL细胞株中CD99表达，能否使其H／RS细胞特性消失或改变，回复到B淋巴瘤样细胞状态呢?
鉴于此，本研究在验证了人HL细胞株L428细胞mic2／CD99基因表达缺失的基础上，利用分子克隆技术构建了pcDNA3.1(+)-CD99真核表达载体，经过稳定转染和克隆筛选，获得L428细胞mic2／CD99基因表达的L428-CD99克隆株，连续观察该株细胞中H／RS细胞的形态及表型改变，构建L428-CD99细胞模型，为进一步研究HL中H／RS细胞的起源及形成的分子机理奠定了的一定的实验基础。
目的
1．明确mic2／CD99在人HL细胞株和cHL组织中H／RS细胞的表达情况。
2．构建mic2／CD99基因的真核表达载体。
3．构建L428-CD99细胞模型并进行初步鉴定。
方法
1．设计寡核苷酸探针，采用分子原位杂交和免疫组化技术检测mic2／CD99在HL细胞株L428中及人15例cHL组织中H／RS细胞的表达。
2．设计mic2／CD99基因特异性PCR引物，采用RT-PCR法从T淋巴瘤细胞株Jurkat细胞总RNA中获取mic2／CD99基因全长cDNA，克隆入T载体，筛选阳性克隆、酶切鉴定并经序列测定；设计含酶切位点的PCR引物，PCR获取mic2／CD99基因表达编码区，用限制性内切酶KpnⅠ和ApaⅠ双酶切取所需目的片段，利用分子克隆技术与pcDNA3.1(+)质粒重组，对产生的重组子进行PCR、双酶切鉴定，并经过测序证实。
3．经过稳定转染和G418克隆筛选，上调L428细胞mic2／CD99基因，用软琼脂克隆形成法和96孔板有限稀释法筛选L428-CD99单克隆，并扩增培养该克隆，建立细胞模型，命名为L428-CD99。
4．应用分子生物学技术检测L428-CD99细胞基因组与pcDNA3.1(+)-CD99载体整合情况；光镜下观察L428-CD99细胞形态特征；光镜下测试网格计数法动态监测由L428的H／RS细胞(直径=25um)转化为L428-CD99细胞的转化率；免疫组化检测CD99、CD30在L428-CD99和L428细胞中的表达；流式细胞术检测L428-CD99、L428细胞及人B淋巴瘤细胞株BJAB中CD30和CD15的表达。
结果
1．人HL细胞株和cHL组织中H／RS细胞mic2／CD99基因检测
人HL细胞株和cHL组织H／RS细胞的mic2／CD99基因mRNA及蛋白(14／15，93.3％)呈阴性表达。
2．mic2／CD99真核表达载体构建
RT-PCR法获得mic2／CD99基因全长cDNA序列，DNA测序的结果与GenBank提供的已知序列(NM-002414)完全一致；重组质粒pcDNA3.1(+)-CD99中的插入片段经DNA测序后与GenBank中mic2／CD99基因相应序列比较100％同源。成功构建了pcDNA3.1(+)-CD99真核表达载体。
3．L428-CD99细胞模型的建立及初步鉴定
利用脂质体转染L428细胞，经G418克隆筛选10d后停药，复苏培养10d后形成L428-CD99单克隆，扩增培养该克隆6-7d，出现明显形念改变，细胞体积变小，细胞核变小，本研究将转型的细胞命名为L428-CD99细胞。定期收获实验组(L428-CD99)和对照组(L428)细胞，检测转染质粒与细胞基因组整合情况，电泳可见基因片段558bp，证实载体已转入细胞，并整合到基因组。细胞计数结果显示，接种96孔板48h后，对照组(L428)中的大细胞或H／RS样细胞(直径=25μm)比例为(10.17±0.0194)％，实验组(L428-CD99)细胞中H／RS样细胞的比例为(3.33±0.0103)％，接种96孔板96h后，L428细胞中的大细胞比例为(11.50±0.0339)％，L428-CD99细胞中H／RS样细胞比例为(3.67±0.0126)％。绘制两组细胞中H／RS细胞生长曲线，经统计分析，L428-CD99细胞中H／RS样细胞与对照组细胞L428相比有统计学差异(P＜0.01)，大H／RS样细胞在L428细胞明显高于L428-CD99细胞。细胞片免疫组化实验显示，CD30蛋白在L428的H／RS细胞胞膜中呈阳性表达，而在L428-CD99细胞的H／RS细胞中呈阴性表达；流式细胞仪检测L428-CD99、L428、BJAB细胞株CD30和CD15的表达，阳性表达率分别为3.17％、55.24％、4.64％；检测CD15的表达，阳性表达率分别为4.15％、12.01％、3.44％。
结论
1．人HL细胞株L428和cHL组织H／RS细胞中mic2／CD99在mRNA水平和蛋白水平均为低表达，CD99低表达或表达缺失或可作为H／RS细胞表型的一个特点。
2．上调人HL细胞株L428细胞mic2／CD99表达，可以诱导H／RS细胞形态、免疫表型和生物学特性发生改变或消失。
创新之处
1．证实人HL细胞株L428和cHL组织中H／RS细胞mic2／CD99的mRNA和蛋白呈低表达，提出CD99低表达或表达缺失或可作为H／RS细胞表型的一个特点。
2．构建了L428-CD99细胞模型，发现mic2／CD99可诱导人HL细胞株L428中H／RS细胞发生转型。
Introduction
Recently, considerable progresses have been made in the study of the etiology,morphology, and the molecular mechanisms of their growth and regulation of H/RScells. Many researches have showed that majority of H/RS cells are originated from"defect B lymphocytes", and the expression of LMP-1, lost expression of CD99protein and lasting activation of NF-κB are the three key elements of H/RS cellsformation. A recent ground breaking discovery has drawn the public attentions to thefact that the originations of H/RS cells are closely related to the lost expression ofCD99 protein. Some recent researches have reported that the antisense CD99transfected to Human B lymphocytes BJAB and IM9 to knockdown the expression ofCD99 can result in classic characteristics of H/RS cells similar to the ones in thelymph nodes of a HL patient, showing "mirror image cell", which over express CD30and CD15 protein. When CD99 gene was transfected into prior mentioned H/RS-likecells, these cells will return to the original phenotype of the B lymphoma. Thisindicates that the origin of H/RS cells, the morphogenesis and change ofimmunophenotype of H/RS cells is related to the lost expression of CD99 gene. Someresearches showed that down-regulation the expression of CD99 can result in thetransformation of H/RS-like cells.
Human CD99 (mic2) is a type of cellular transmembrane protein with 32kDa. Itscoding gene is located at the site of Xp22.32-pter and Yp11-pter in the PAR zones ofthe X and Y chromosome short chains. Genomic sequencing analysis indicated thatPAR is the potential oncogene for HL, participating in the differentiation ofhemocytes. Our previous study showed that using RNAi technology to silence themCD99L2 gene from A20 cells, which result in the transformation of H/RS-like cells,and confirm its immunophenotype characteristic and biological features. So howabout up-regulate CD99 expression in human HL cell line to make these cells returnto the original phenotype of the B lymphoma?
We detected the expression of mic2/CD99 gene was negetive in H/RS cells ofhuman HL cell line L428, and constructed the eukaryotic expressing vectorpcDNA3.1(+)-CD99. The L428-CD99 cell clone expressing mic2/CD99 gene wasgain by stable transfection and clone selected. We observed the morphology and theimmunophenotype of H/RS cells of L428-CD99, and constructed the L428-CD99 cellmodel. It's the basic of further to studying the etiology and the molecular mechanismsof H/RS cells of HL.
Objectives
1. To make clear CD99 mRNA and protein level expression of the H/RS cells of theHuman HL cell line and the cHL tissue sample.
2. Construct the mic2/CD99 gene eukaryotic expressing vector.
3. Constrsct the L428-CD99 cell model and confirm its character.
Method
1. Design oligo-nucletide probe, and detect CD99 expression in H/RS cells of HLcell line and 15 samples of cHL tissue by molecular hybridization in situ andimmunohistochemistry.
2. Design CD99 gene specific PCR primer, and acquire CD99 cDNA from T lymphoma cell line Jurkat total RNA by RT-PCR, clone it into T vector, get positiveclone, make enzyme cleaving and DNA sequencing confirmation. Design PCR primerwith enzyme cleavage locus, get CD99 gene expression frame by PCR, then combineto the vector pcDNA3.1 (+). The sequence of the PCR product was confirmed byenzyme cleaving and DNA sequencing analysis.
3. Stable transfer and selected by G418, up-regulation of CD99 gene in L428, andget the monoclone by using soft agar clone formation protocol and 96 well platelimiting dilution assay, proliferate this clone and named the result cell modelL428-CD99.
4. Examine the integrated status of L428-CD99 clone and pcDNA3.1 (+)-CD99vector by using molecular biology technique. Observe the morphologicalcharacteristics of L428-CD99 cells under light microscope. Evaluate transformationrate of H/RS cells of L428 (diameter=25μm) transformed to L428-CD99 by netcounting method. Detect the CD99,CD30 protein expression in L428-CD99 clone andL428 by immunohistochemistry. The expression of CD30 and CD15 of L428-CD99、L428、BJAB cell line was tested with flow cytometer.
Result
1. CD99 expression in H/RS cells of HL cell lines and cHL tissue sample:
CD99 expression was negative in H/RS cells of HL cell line and cHL tissuesample (14/15,93.3%) at mRNA and protein level.
2. Construction of CD99 eukaryotic expression vector:
Full length cDNA of CD99 was acquired by RT-PCR and gained correctsequencing confirmation. The sequence was totally consistent with the data ofGenBank (NM-002414) by DNA sequencing analysis. The sequence of therecombination plasmid pcDNA3.1 (+)-CD99 was totally consistent with the data ofGenBank through DNA sequencing analysis. Eukaryotic expression vector pcDNA3.1(+)-CD99 was successfully constructed.
3. Construst the L428-CD99 cell model and confirm its chatacter:
Transfer the L428 cells by liposome and the positive cell clones were selected andobtained by G418 after 10 days. Obvious morphological changes, including volumeand nucleus decrease were observed after 6-7 days. We named this cell clone asL428-CD99. 558 bp fragments were detected in L428-CD99 group by electrophoresis,which served as an evidence of successfully transfection. Cell counting resultsshowed that after 48 h of inoculation in 96 well plate, rate of H/RS-like cells (over 25μm in diameter) in control group L428 was (10.17±0.0194)%, and itwas(3.33±0.0103)% in L428-CD99 group. H/RS-like cells in L428 cells after 96 hinoculation were (11.50±0.0339)%, H/RS-like cells in L428-CD99 were(3.67±0.0126)%. H/RS-like cells in L428 group were significantly more than inL428-CD99 group (P＜0.01). Result of immunohistochemistry showed that CD30protein expression was positive in H/RS cells of L428, and was negative in H/RScells of L428-CD99 cells and CD99 protein expression was positive in H/RS cells ofL428 and was negative in L428-CD99. The expression of CD30 and CD15 of celllines were tested with flow cytometer showed that the expression of CD30 ofL428-CD99 and L428 was respectively 3.17% and 55.24%. The expression of CD30of BJAB cell line was 4.64%. And the expression of CD15 of L428-CD99 and L428was respectively 4.15% and 12.01%, and the expression of CD15 of BJAB was3.44%.
Conclusion
1. CD99 expression in H/RS cells of Human HL cell line L428 and cHL tissuesamples were both negative at mRNA level and protein level, which may served asone of its phenotype characteristics.
2. Up-regulation CD99 gene expression in human HL cell line L428 will result inthe changes of the morphology, immunophenotype and biological features of H/RScells, which return to the characteristics of B lymphocytes.
Original ideas of the study
1. Confirmed CD99 expression at mRNA level and protein level was negative orlow in H/RS cells of human HL cell line L428 and cHL tissue samples, indicatedthat CD99 expression was negetive or low may served as one of its phenotypecharacteristics.
2. Construct the L428-CD99 cell modle. Observed that mic2/CD99 gene maytransform H/RS cells of human HL cell line L428 into B lymphoma-like cells.
引文
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[1] Kim SH, Choi EY, Shin YK, et al. Generation of cells with Hodgkin's and Reed-Steinberg phenotype through downregulation of CD99(Mic2)[J]. Blood, 1998, 92: 4287-4295.
[2] 李先茂，李燕，赵彤，等．霍奇金淋巴瘤CD99基因表达缺失的意义[J]．第四军医大学学报，2004；25(23)：2136-2137
[3] Kim SH, Shin YK, Lee IS, et al .Viral latent membrane protein 1(LMP-1)induced CD99 down-regulation in B cells leads to the generation of cells with Hodgkin's and Reed-Steinberg phenotype [J]. Blood, 2000, 95(1):294-300
[1] Kim SH, Choi EY, Shin YK, et al. Generation of cells with Hodgkin's and Reed-Stemberg phenotype through downregulation of CD99 (Mic2) [J]. Blood. 1998, 92(11):4287-95.
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