Degranulation influences heparin-associated inhibition of RT-PCR in human lung mast cells.
Degranulation influences heparin-associated inhibition of RT-PCR in human lung mast cells.
复制标题
脱颗粒影响人肺肥大细胞中肝素相关的 RT-PCR 抑制。
DOI：
10.1046/j.1365-2222.2001.01196.x
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Schulman,ES
中科院分区：
文献类型：
--
作者：
Glaum,MC;Wang,Y;Raible,DG;Schulman,ES
背景人类肺肥大细胞（HLMC）基因表达的研究受到了限制，因为不能可靠地检测到少量被肝素污染的肥大细胞RNA的mRNA转录本。由于肝素在肥大细胞内是颗粒相关的，我们研究了脱粒在改变这种蛋白多糖抑制HLMC RNA逆转录聚合酶链反应（RT-PCR）的内在能力中的作用。我们还探索了RNA分离的替代手段，以消除初级heparincontamination.MethodsPurified HLMC（纯度> 90%）挑战2小时，缓冲液，抗IgE（3 μg/mL）和/或离子载体A23187（100 ng/mL）或佛波醇12-肉豆蔻酸酯13-乙酸酯（50 ng/mL）。 使用分光荧光测定法测量组胺释放。激发后，通过苯酚-氯仿提取或硝酸纤维素旋转柱分离RNA。平行样品用肝素酶处理或在逆转录前置于冰上2小时。 使用管家基因特异性引物进行PCR，甘油醛-3-磷酸脱氢酶（GAPDH）。结果在五项研究中，GAPDH条带在来自脱颗粒肥大细胞的总细胞RNA（tcRNA）中的强度高于非脱颗粒肥大细胞。然而，当检查来自相同肥大细胞样品的肝素酶处理的tcRNA时，脱颗粒细胞和非脱颗粒细胞之间GAPDH条带的强度标准化。当比较柱纯化tcRNA的平行样品时，随后用肝素酶处理样品对GAPDH的检测没有影响，表明该技术有效地去除了内源性肝素。此外，在静息与脱颗粒肥大细胞（n= 3）中检测到的GAPDH信号中没有观察到变化。结论脱颗粒影响HLMC中肝素相关的RT-PCR抑制程度，并且旋转柱纯化tcRNA是肝素酶预处理的一种省时、有效的替代方法。
BackgroundThe study of gene expression from human lung mast cells (HLMC) has been limited by the ability to reliably detect mRNA transcripts from scant quantities of mast cell RNA contaminated with heparin.ObjectiveAs heparin is granule‐associated within the mast cell, we examined the role of degranulation in altering the intrinsic ability of this proteoglycan to inhibit reverse transcription polymerase chain reaction (RT‐PCR) from HLMC RNA. We also explored alternative means of RNA isolation to eliminate primary heparin contamination.MethodsPurified HLMC (> 90% pure) were challenged for 2 h with buffer, anti‐IgE (3 µg/mL) and/or ionophore A23187 (100 ng/mL) or phorbol 12‐myristate 13‐acetate (50 ng/mL). Histamine release was measured using a spectroflourometric assay. Following challenge, RNA was isolated by either phenol‐chloroform extraction or by nitrocellulose spin column. Parallel samples were either treated with heparinase or placed on ice for 2 h prior to reverse transcription. PCR was performed using primers specific for the housekeeping gene, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH).ResultsIn each of five studies, GAPDH bands were detected at greater intensity in total cellular RNA (tcRNA) derived from degranulated than from non‐degranulated mast cells. However, when examining heparinase‐treated tcRNA from the same mast cell samples, the intensity of GAPDH bands normalized between degranulated and non‐degranulated cells. When comparing parallel samples of column‐purified tcRNA, subsequent treatment of samples with heparinase had no effect on the detection of GAPDH, indicating that endogenous heparin was effectively removed by the technique. Moreover, no variability was noted in GAPDH signal detected from resting vs. degranulated mast cells (n= 3).ConclusionsDegranulation influences the degree of heparin‐associated inhibition of RT‐PCR in HLMC, and that spin column purification of tcRNA is a time‐saving, effective alternative to heparinase pre‐treatment.