Cyr61/CCN1在银屑病炎性微环境中的功能及凉血潜阳法调控研究

Based on the clinical experience of the XiaShi surgery treatment of skin diseases and the previous studies, it's confirmed by research group that cooling blood and restrain Yang treatment could inhibit keratinocyte dysplasia. A recent study found that Cyr61 is the key factor to promote psoriatic epidermal proliferation and inflammatory infiltration in psoriatic inflammatory microenvironment. On this basis, we try to discussed cooling blood and restrain Yang treatment's cell biological mechanisms on psoriasis vulgaris from the Cyr61 regulation of KC inflammatory microenvironment . The project targeted to Imiquimod-induced mouse model of psoriasis and psoriatic patients and normal keratinocyte, using shRNA Lentiviral Particles and the expression method of Anti-Cyr61 antibody interference in Cyr61 to studied on the function of the Cyr61 in the psoriatic inflammatory microenvironment and to explain the influnce carryed by the cooling blood and restrain Yang treatment to regulate the biological behavior of cell proliferation and cell cycle as well cell apoptosis . By using the test card method of TCM theory to proofed and disproofed of observing treatment effect on the "blood heat syndrome patients with psoriasis vulgaris and to conducted correlation study between regulating Cyr61 protein expression and inflammatory microenvironment in patients with psoriasis. From this way to investigate inherent laws of the development process regulated by Cyr61 that keratinocyte inflammatory microenvironment involved in psoriasis "blood heat syndrome" and molecular biological mechanisms of cooling blood and restrain Yang treatment in psoriatic inflammatory microenvironment.

基于夏氏外科"凉血潜阳法"治疗银屑病临床经验，课题组已证明凉血潜阳法明显抑制KC异常增生，近期研究发现Cyr61在银屑病炎性微环境中,是促进银屑病表皮过度增殖及炎症浸润作用的关键因子。在此基础上，我们试图从Cyr61调控KC炎性微环境探讨凉血潜阳法对寻常型银屑病作用细胞生物学机制。项目以咪喹莫特诱导小鼠银屑病模型、正常人和银屑病患者KC为研究对象，采用shRNA慢病毒颗粒及与Anti-Cyr61抗体干扰Cyr61表达方法，研究Cyr61在银屑病炎性微环境中的功能，以期阐释凉血潜阳法调控KC增殖、周期及凋亡等生物学行为影响。并运用中医方药理论中以方测证方法对寻常型银屑病"血热证"患者治疗效应观察印证和反证，并与调控患者Cyr61蛋白表达及炎性微环境进行相关性研究，探讨Cyr61调控KC炎性微环境参与银屑病"血热证"发生发展过程中内在规律，以及凉血潜阳法对银屑病炎性微环境作用细胞分子生物学机制

基于夏氏外科“凉血潜阳法”治疗银屑病临床经验，课题组已证明凉血潜阳法明显抑制KC异常增生，近期研究发现Cyr61在银屑病炎性微环境中,是促进银屑病表皮过度增殖及炎症浸润作用的关键因子。在此基础上，我们进一步从Cyr61调控KC炎性微环境探讨凉血潜阳法对寻常型银屑病作用细胞生物学机制。体內试验部分，临床上我们以正常人、寻常型银屑病血热证患者、芩珠凉血方治疗愈显组患者、芩珠凉血方治疗无效组患者为研究对象；动物模型以咪喹莫特诱导的银屑病模型为研究对象，运用组织病理；皮损严重程度评分及耳朵肿胀程度变化，流式细胞术比较芩珠凉血方对皮肤组织中免疫细胞的影响; Real-time PCR检测该方对皮肤组织中 Cyr61、IL-17、IL-23、TNF-α、IL-6、IL-21 和 IFN-γ等相关细胞因子影响；免疫组化及Western blot比较Cyr61蛋白变化，以验证芩珠凉血方治疗银屑病的有效性及对其Cyr61影响。实验结果表明芩珠凉血方可以改善皮肤炎症及下调Cyr61的表达，提示芩珠凉血方改善银屑病炎症可能与Cyr61的下调有关。为了进一步探讨芩珠凉血方是否直接下调Cyr61，我们通过外源性注射IL-17入咪喹莫特诱导小鼠双耳，经过芩珠凉方干预，比较小鼠耳朵肿胀变化；Real-time PCR技术检测皮损中Cyr61表达变化；流式细胞术检测皮损中炎症细胞的变化。结果发现外源性IL-17加入后，芩珠组小鼠的炎症反应与对照组无明显差异，说明凉血潜阳方可能通过下调IL-17，进而下调Cyr61，最终发挥抗炎作用。为了明确芩珠凉血方下调IL-17的靶细胞。运用流式细胞术，发现该方可抑制真皮γδΤ细胞分泌IL-17，对Th17无影响。体外实验以小鼠角质形成细胞(PAM212)为研究对象，通过外源性IL-17刺激，Real-time PCR检测细胞中Cyr61表达，结果发现含药血清可降低Cyr61表达，外源性IL-17加入后，两组间则无明显差异。再次验证凉血潜阳方可能通过下调IL-17，进而下调Cyr61。本研究得出结论：凉血潜阳法通过抑制真皮γδΤ细胞分泌IL-17下调Cyr61来改善小鼠银屑病样皮炎。该成果丰富和完善目前指导银屑病临床诊疗的“血分论治”理论，同时对中医药辨证治疗银屑病的作用靶点，中医临床客观诊疗具有积极意义。