Knuppe Molecular Urology Laboratory-Department of Urology, School of Medicine, University of California, San Francisco, CA 94143, USA.
J Sex Med. 2011 Feb;8(2):437-46. doi: 10.1111/j.1743-6109.2010.02128.x. Epub 2010 Nov 29.
Previously we reported that paracrine actions likely mediated the therapeutic effects of adipose tissue-derived stem cells (ADSCs) on a rat model of cavernous nerve (CN) injury.
To identify potential neurotrophic factors in ADSC's secretion, test the most promising one, and identify the molecular mechanism of its neurotrophic action.
Rat major pelvic ganglia (MPG) were cultured in conditioned media of ADSC and penile smooth muscle cells (PSMCs). Cytokine expression in these two media was probed with a cytokine antibody array. CXCL5 cytokine was quantified in these two media by enzyme-linked immunosorbent assay (ELISA). Activation of Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) by CXCL5 was tested in neuroblastoma cell lines BE(2)C and SH-SY5Y as well as in Schwann cell line RT4-D6P2T by Western blot. Involvement of CXCL5 and JAK/STAT in ADSC-conditioned medium's neurotrophic effects was confirmed with anti-CXCL5 antibody and JAK inhibitor AG490, respectively.
Neurotrophic effects of ADSC and PSMC-conditioned media were quantified by measuring neurite length in MPG cultures. Secretion of CXCL5 in these two media was quantified by ELISA. Activation of JAK/STAT by CXCL5 was quantified by densitometry on Western blots for STAT1 and STAT3 phosphorylation.
MPG neurite length was significantly longer in ADSC than in PSMC-conditioned medium. CXCL5 was secreted eight times higher in ADSC than in PSMC-conditioned medium. Anti-CXCL5 antibody blocked the neurotrophic effects of ADSC-conditioned medium. CXCL5 activated JAK/STAT concentration-dependently from 0 to 50 ng/mL in RT4-D6P2T Schwann cells. At 50 ng/mL, CXCL5 activated JAK/STAT time-dependently, peaking at 45 minutes. AG490 blocked these activities as well as the neurotrophic effects of ADSC-conditioned medium.
CXCL5 was secreted by ADSC at a high level, promoted MPG neurite growth, and activated JAK/STAT in Schwann cells. CXCL5 may contribute to ADSC's therapeutic efficacy on CN injury-induced ED.
我们之前曾报道过旁分泌作用可能介导了脂肪组织源性干细胞（ADSCs）对大鼠海绵体神经（CN）损伤模型的治疗作用。
鉴定 ADSC 分泌液中的潜在神经营养因子，测试最有前途的一种，并鉴定其神经营养作用的分子机制。
在 ADSC 和阴茎平滑肌细胞（PSMC）的条件培养基中培养大鼠主要盆腔神经节（MPG）。用细胞因子抗体阵列探测这两种培养基中的细胞因子表达。通过酶联免疫吸附试验（ELISA）定量检测这两种培养基中的 CXCL5 细胞因子。通过 Western blot 测试 CXCL5 在神经母细胞瘤细胞系 BE(2)C 和 SH-SY5Y 以及施万细胞系 RT4-D6P2T 中对 Janus 激酶/信号转导和转录激活因子（JAK/STAT）的激活作用。用抗 CXCL5 抗体和 JAK 抑制剂 AG490 分别证实 CXCL5 和 JAK/STAT 在 ADSC 条件培养基中的神经营养作用。
通过测量 MPG 培养物中的神经突长度来定量评估 ADSC 和 PSMC 条件培养基的神经营养作用。ELISA 定量检测这两种培养基中 CXCL5 的分泌。通过 densitometry 对 Western blot 中 STAT1 和 STAT3 磷酸化的定量来评估 CXCL5 对 JAK/STAT 的激活作用。
MPG 神经突长度在 ADSC 条件培养基中明显长于 PSMC 条件培养基。ADSC 条件培养基中 CXCL5 的分泌量比 PSMC 条件培养基高 8 倍。抗 CXCL5 抗体阻断了 ADSC 条件培养基的神经营养作用。CXCL5 在浓度为 0 至 50ng/ml 时，以浓度依赖的方式激活 RT4-D6P2T 施万细胞中的 JAK/STAT，在 50ng/ml 时，CXCL5 时间依赖性地激活 JAK/STAT，在 45 分钟时达到峰值。AG490 阻断了这些活性以及 ADSC 条件培养基的神经营养作用。
ADSC 大量分泌 CXCL5，促进 MPG 神经突生长，并激活施万细胞中的 JAK/STAT。CXCL5 可能有助于 ADSC 对 CN 损伤诱导的 ED 的治疗效果。
