IL-22在Behcet病及Vogt-小柳原田綜合征中的表達(dá)及其影響因素的研究
發(fā)布時(shí)間：2019-07-06 09:59
【摘要】：第一部分 IL-22在Behcet病中的表達(dá)及其意義 1.目的 檢測(cè)IL-22在Behcet病患者外周血及皮損中的表達(dá)情況，分析其與疾病活動(dòng)及臨床癥狀的相關(guān)性。 2.方法： 2.1密度梯度法分離患者及正常人外周單個(gè)核細(xì)胞（PBMCs），磁珠分選CD4~+T細(xì)胞，加入抗CD3和CD28抗體刺激行細(xì)胞培養(yǎng)。 2.2采用酶聯(lián)免疫吸附法（ELISA）檢測(cè)患者及健康對(duì)照者血清、PBMCs及CD4~+T細(xì)胞培養(yǎng)上清中IL-22的含量。 2.3流式細(xì)胞儀檢測(cè)外周血中IL-22~+CD4~+T細(xì)胞、IL-22~+CD8~+T細(xì)胞的比例，及IL-22、IL-17共表達(dá)的情況。 2.4Real-time PCR法檢測(cè)結(jié)節(jié)性紅斑中IL-22mRNA的表達(dá)。 2.5依據(jù)PBMC培養(yǎng)上清中IL-22的含量將患者分為“IL-22正常組”與“IL-22高表達(dá)組”，分別與臨床特點(diǎn)，血清標(biāo)志物進(jìn)行統(tǒng)計(jì)學(xué)分析。 3.結(jié)果 3.1IL-22在活動(dòng)期BD患者，靜止期BD患者，急性特發(fā)性前葡萄膜炎患者及正常人的血清水平?jīng)]有顯著性差異。 3.2在活動(dòng)期Behcet病患者中， PBMCs細(xì)胞培養(yǎng)上清中的IL-22含量顯著高于靜止期Behcet患者，急性特發(fā)性前葡萄膜炎患者及正常人。同樣，在活動(dòng)期Behcet病患者中， CD4~+T細(xì)胞培養(yǎng)上清中的IL-22含量顯著高于靜止期Behcet患者及正常人。 3.3在活動(dòng)期Behcet病患者中，IL-22~+CD4~+T細(xì)胞的比例顯著高于靜止期Behcet患者及正常人。同時(shí)，IL-22,IL-17雙陽性的CD4~+T細(xì)胞的比例在活動(dòng)期BD患者也顯著升高。BD患者及正常人均只有小部分IL-22~+CD4~+T細(xì)胞（21%-27%）共表達(dá)IL-17,而大部分的IL-22~+CD~+T細(xì)胞是IL-17陰性的。 3.4結(jié)節(jié)性紅斑中IL-22的mRNA表達(dá)顯著高于正常皮膚。 3.5BD患者PBMCs培養(yǎng)上清中升高的IL-22與前房細(xì)胞，視網(wǎng)膜血管炎及結(jié)節(jié)性紅斑正相關(guān)。 4.結(jié)論 實(shí)驗(yàn)結(jié)果顯示IL-22的表達(dá)與Behcet病的活動(dòng)性及小血管炎相關(guān)，提示其可能參與了Behcet病的發(fā)病。 第二部分 Behcet病中IL-23，地塞米松及環(huán)孢素A對(duì)IL-22表達(dá)的影響 1.目的 研究BD患者中，IL-23是否可誘導(dǎo)IL-22分泌，地塞米松及環(huán)孢素A是否可抑制IL-22的分泌。 2.方法 2.1分離BD患者及在正常人PBMCs，磁珠分選CD4~+T細(xì)胞，進(jìn)行不同條件下細(xì)胞培養(yǎng)。 2.2CD4~+T細(xì)胞分4組。1組為基礎(chǔ)培養(yǎng)組，2組加入抗C D3和C D28抗體，，3組加抗C D3和C D28抗體及r h I L-23，4組為只加入rhIL-23組。 2.3PBMCs細(xì)胞分6組。1組為基礎(chǔ)培養(yǎng)基，2組每孔加入抗C D3和C D28抗體刺激，3組在B組基礎(chǔ)上加入不同濃度的地塞米松，4組在B組基礎(chǔ)上加入不同濃度的環(huán)孢素A。5組在C組基礎(chǔ)上加入rhIL-23。6組在D組基礎(chǔ)上加入rhIL-23。 3.結(jié)果 3.1IL-23可以誘導(dǎo)BD患者和正常人活化的CD4~+T細(xì)胞分泌IL-22顯著增加。并且在活動(dòng)期的BD患者中，rIL-23誘導(dǎo)CD4~+T細(xì)胞產(chǎn)生的IL-22顯著高于靜止期BD患者和正常人。 3.2地塞米松和環(huán)孢素A在體外均可抑制患者及正常人PBMCs分泌IL-22，并且在一定濃度范圍內(nèi)，這種抑制作用呈劑量依賴性。100ng/ml地塞米松可抑制70%IL-22的產(chǎn)生，10ng/ml環(huán)孢素A可抑制80%IL-22的產(chǎn)生。 3.3PBMCs加入地塞米松或環(huán)孢素A培養(yǎng)后，活動(dòng)期BD患者和正常人rIL-23誘導(dǎo)PBMCs分泌IL-22的能力均明顯受到抑制。 4．結(jié)論 活動(dòng)期BD患者中，IL-22的表達(dá)增加可能由于其上游的IL-23誘導(dǎo)分泌所致，提示BD中IL-23/IL-22通路的存在。地塞米松及環(huán)孢素A可抑制IL-22的產(chǎn)生。 第三部分 IL-22在Vogt-小柳原田綜合征中的表達(dá)及其意義 1.目的 檢測(cè)IL-22在VKH患者外周血中的表達(dá)情況，分析其與疾病活動(dòng)的相關(guān)性。 2.方法： 2.1采用酶聯(lián)免疫吸附法（ELISA）檢測(cè)患者及健康對(duì)照者血清、PBMCs及CD4~+T細(xì)胞的培養(yǎng)上清中IL-22的含量 2.2流式細(xì)胞儀檢測(cè)VKH患者及正常人外周血中IL-22~+CD4~+/CD8~+T細(xì)胞，IL-22~+IL-17~+CD4~+T的比例。 2.3依據(jù)VKH患者PBMC培養(yǎng)上清中IL-22的含量將患者分為“IL-22正常組”與“IL-22高表達(dá)組”，分別與臨床特點(diǎn)，血清標(biāo)志物進(jìn)行統(tǒng)計(jì)學(xué)分析。 3.結(jié)果 3.1IL-22在活動(dòng)期VKH患者，靜止期VKH患者及正常人的血清水平?jīng)]有顯著性差異。 3.2在PBMCs及CD4~+T細(xì)胞培養(yǎng)上清中的IL-22含量，活動(dòng)期VKH病患者中顯著高于靜止期VHK患者及正常人。 3.3IL-22~+CD4~+T細(xì)胞的比例，活動(dòng)期VKH病患者顯著高于靜止期VKH患者及正常人。IL-22~+CD8~+T細(xì)胞的比例在三者之間沒有顯著性差異。在VKH患者及正常人中，CD4~+T細(xì)胞均是分泌IL-22的主要細(xì)胞。IL-22,IL-17雙陽性的CD4~+T細(xì)胞的比例在活動(dòng)期VKH患者中也顯著升高。 3.4VKH患者PBMCs培養(yǎng)上清中升高的IL-22與羊脂狀KP，前房細(xì)胞成正相關(guān)。 4.結(jié)論 實(shí)驗(yàn)結(jié)果顯示IL-22的表達(dá)與VKH綜合征的活動(dòng)性正相關(guān)，提示其可能參與了VKH綜合征的發(fā)病。 第四部分 VKH綜合征中IL-23，地塞米松及環(huán)孢素A對(duì)IL-22表達(dá)的影響 1.目的 研究VKH患者中，IL-23是否可誘導(dǎo)IL-22分泌，地塞米松及環(huán)孢素A是否可抑制IL-22的分泌。 2.方法 2.1分離VKH患者及在正常人PBMCs，磁珠分選CD4~+T細(xì)胞，進(jìn)行不同條件下細(xì)胞培養(yǎng)。 2.2CD4~+T細(xì)胞分4組。1組為基礎(chǔ)培養(yǎng)組，2組加入抗C D3和C D28抗體，3組加抗C D3和C D28抗體及r h I L-23，4組只加rhIL-23。 2.3PBMCs細(xì)胞分5組。1組為基礎(chǔ)培養(yǎng)基，2組每孔加入抗C D3和C D28抗體刺激，3組在B組基礎(chǔ)上加入不同濃度的地塞米松，4組在B組基礎(chǔ)上加入不同濃度的環(huán)孢素A。5組在B組基礎(chǔ)上加入10ng/ml環(huán)孢素A及100ng/ml地塞米松。 3.結(jié)果 3.1IL-23可以誘導(dǎo)VKH患者和正常人抗CD3抗體和抗CD28抗體刺激的CD4~+T細(xì)胞分泌IL-22顯著增加。在活動(dòng)期的VKH患者中，rIL-23誘導(dǎo)CD4~+T細(xì)胞產(chǎn)生的IL-22顯著高于靜止期患者和正常人。 3.2在體外的PBMCs培養(yǎng)體系中，地塞米松和環(huán)孢素A均可抑制VKH患者及正常人PBMCs分泌IL-22，這種抑制作用呈劑量依賴性。兩種藥物聯(lián)合后抑制效果更加明顯，可抑制95%的IL-22分泌。 4.結(jié)論 活動(dòng)期VKH患者中，IL-22的表達(dá)增加可能由于其上游的IL-23誘導(dǎo)分泌所致，提示VKH中IL-23/IL-22通路的存在。地塞米松及環(huán)孢素A可抑制IL-22的產(chǎn)生。
文內(nèi)圖片：
圖片說明：活動(dòng)期BD患者（n=10）、靜止期BD患者（n=8）及正常人(n=10)外周血
[Abstract]:the first part The expression of IL-22 in Behcet's disease and its expression meaning Objective To investigate the expression of IL-22 in peripheral blood and skin lesions of patients with Behcet's disease. the correlation of the shape .2. Method: 2.1 The peripheral mononuclear cells (PBMCs) of the patients and the normal persons were isolated by density gradient method, CD4 ~ + T cells were sorted by magnetic beads, anti-CD3 and CD28 were added. Antibody-stimulated line cell culture 2.2.2 The serum, PBMCs and CD4 ~ + T cell culture of patients and healthy controls were detected by enzyme-linked immunosorbent assay (ELISA). The ratio of IL-22 ~ + CD4 ~ + T cells, IL-22 ~ + CD8 ~ + T cells and IL-2 in peripheral blood were detected by flow cytometry. 2. The co-expression of IL-17. The expression of IL-22 mRNA in erythema. 2.5 The patients were divided into "IL-22 normal group" and "IL-22 high expression group" according to the content of IL-22 in the supernatant of PBMC culture. Clinical features 3. Results 3.1 IL-22 in active BD patients, patients with BD, acute idiopathic preglucomannan There was no significant difference in serum levels in patients with mucositis and normal controls. 3.2 The level of IL-22 in the supernatant of the PBMCs was significantly higher in patients with active Behcet's disease than in the rest period Hcet, patients with acute idiopathic anterior uveitis, and normal subjects. Similarly, in patients with active Behcet, IL-in the supernatant of CD4 + T cell culture The content of IL-22 ~ + CD4 ~ + in patients with active Behcet was significantly higher than that of Behcet's and normal subjects. The proportion of T cells was significantly higher than that of Behcet's and normal controls. At the same time, IL-22 and IL-17 were positive. The proportion of CD4 ~ + T cells increased significantly in active BD patients. Only small part of IL-22 ~ + CD4 ~ + T cells (21% -27%) of BD patients and normal individuals co-express IL-17, and most of them The IL-22 ~ + CD ~ + T cells were negative for IL-17. The expression of IL-22 in nodular erythema was significantly higher than that of normal skin. raised I The results showed that the expression of IL-22 was similar to that of Behc. et-disease Activity and associated vasculitis suggest that it may be involved in the onset of Behcet's disease. part Effect of IL-23, Dexamethasone and Cyclosporin A on the Expression of IL-22 in Behcet's Disease 1. Objective To Study the Effect of IL-23, Dexamethasone and Cyclosporin A on the Expression of IL-22. , IL- 23. Whether IL-22 secretion, dexamethasone and cyclosporin A can be induced to inhibit the secretion of IL-22. 2.1 The patients with BD were isolated from BD and the CD4 ~ + T cells were sorted by magnetic beads, and the cells were cultured under different conditions. 2.2 CD4 ~ + T cells were divided into 4 groups. The anti-C D3 and C D28 antibodies were added to the D3 and C D28, and the anti-C D3 and C D28 antibodies in the 3 groups and the r h I-23 and 4 groups were only added to the rhIL-23 group. to join in The rhIL-23.6 group was added to the group of rhIL-23.6 on the basis of group C, and rhIL-23.6 was added to the group C. Results 3.1 IL-23 could induce a significant increase in the secretion of IL-22 from the activated CD4 + T cells in patients with BD and normal controls. And in the patients with active BD, the IL-22 induced by rIL-23 in the CD4 + T cells was significantly higher than that of the patients in the quiescent period and the normal controls. 3.2.2 The effect of dexamethasone and cyclosporin A in vitro inhibited the secretion of IL-22 from the PBMCs of the patient and the normal human, and the inhibitory effect was in the range of a certain concentration. Reactivity.100 ng/ ml of dexamethasone inhibited the production of 70% IL-22 and 10 ng/ ml of cyclosporine A inhibited the production of 80% IL-22. 3.3 PBMC s Add-in Conclusion: The ability of IL-22 to secrete IL-22 in patients with active BD and normal human rIL-23 was significantly inhibited after the culture of Celecoxib or Cyclosporin A. In active BD patients, the increase in the expression of IL-22 may can be IL-23 induced by the upstream IL-23, suggesting that IL-23 in BD/ IL-2 2. The presence of the pathway. Dexamethasone and cyclosporine A inhibit the production of IL-22. Part III IL-22 Expression of IL-22 in the peripheral blood of patients with VKH and its significance in the analysis of the expression of IL-22 in the peripheral blood of patients with VKH 2. Methods: 2.1 The serum, PBMCs and CD4 ~ + T cells were detected by enzyme-linked immunosorbent assay (ELISA) in the culture of serum, PBMCs and CD4 ~ + T cells. 22. The proportion of IL-22 ~ + CD4 ~ +/ CD8 ~ + T cells, IL-22 ~ + IL-17 ~ + CD4 ~ + T in the peripheral blood of patients with VKH and normal persons was detected by flow cytometry. norm al group" 涓
本文編號(hào)：2510935
文內(nèi)圖片：
圖片說明：活動(dòng)期BD患者（n=10）、靜止期BD患者（n=8）及正常人(n=10)外周血
[Abstract]:the first part The expression of IL-22 in Behcet's disease and its expression meaning Objective To investigate the expression of IL-22 in peripheral blood and skin lesions of patients with Behcet's disease. the correlation of the shape .2. Method: 2.1 The peripheral mononuclear cells (PBMCs) of the patients and the normal persons were isolated by density gradient method, CD4 ~ + T cells were sorted by magnetic beads, anti-CD3 and CD28 were added. Antibody-stimulated line cell culture 2.2.2 The serum, PBMCs and CD4 ~ + T cell culture of patients and healthy controls were detected by enzyme-linked immunosorbent assay (ELISA). The ratio of IL-22 ~ + CD4 ~ + T cells, IL-22 ~ + CD8 ~ + T cells and IL-2 in peripheral blood were detected by flow cytometry. 2. The co-expression of IL-17. The expression of IL-22 mRNA in erythema. 2.5 The patients were divided into "IL-22 normal group" and "IL-22 high expression group" according to the content of IL-22 in the supernatant of PBMC culture. Clinical features 3. Results 3.1 IL-22 in active BD patients, patients with BD, acute idiopathic preglucomannan There was no significant difference in serum levels in patients with mucositis and normal controls. 3.2 The level of IL-22 in the supernatant of the PBMCs was significantly higher in patients with active Behcet's disease than in the rest period Hcet, patients with acute idiopathic anterior uveitis, and normal subjects. Similarly, in patients with active Behcet, IL-in the supernatant of CD4 + T cell culture The content of IL-22 ~ + CD4 ~ + in patients with active Behcet was significantly higher than that of Behcet's and normal subjects. The proportion of T cells was significantly higher than that of Behcet's and normal controls. At the same time, IL-22 and IL-17 were positive. The proportion of CD4 ~ + T cells increased significantly in active BD patients. Only small part of IL-22 ~ + CD4 ~ + T cells (21% -27%) of BD patients and normal individuals co-express IL-17, and most of them The IL-22 ~ + CD ~ + T cells were negative for IL-17. The expression of IL-22 in nodular erythema was significantly higher than that of normal skin. raised I The results showed that the expression of IL-22 was similar to that of Behc. et-disease Activity and associated vasculitis suggest that it may be involved in the onset of Behcet's disease. part Effect of IL-23, Dexamethasone and Cyclosporin A on the Expression of IL-22 in Behcet's Disease 1. Objective To Study the Effect of IL-23, Dexamethasone and Cyclosporin A on the Expression of IL-22. , IL- 23. Whether IL-22 secretion, dexamethasone and cyclosporin A can be induced to inhibit the secretion of IL-22. 2.1 The patients with BD were isolated from BD and the CD4 ~ + T cells were sorted by magnetic beads, and the cells were cultured under different conditions. 2.2 CD4 ~ + T cells were divided into 4 groups. The anti-C D3 and C D28 antibodies were added to the D3 and C D28, and the anti-C D3 and C D28 antibodies in the 3 groups and the r h I-23 and 4 groups were only added to the rhIL-23 group. to join in The rhIL-23.6 group was added to the group of rhIL-23.6 on the basis of group C, and rhIL-23.6 was added to the group C. Results 3.1 IL-23 could induce a significant increase in the secretion of IL-22 from the activated CD4 + T cells in patients with BD and normal controls. And in the patients with active BD, the IL-22 induced by rIL-23 in the CD4 + T cells was significantly higher than that of the patients in the quiescent period and the normal controls. 3.2.2 The effect of dexamethasone and cyclosporin A in vitro inhibited the secretion of IL-22 from the PBMCs of the patient and the normal human, and the inhibitory effect was in the range of a certain concentration. Reactivity.100 ng/ ml of dexamethasone inhibited the production of 70% IL-22 and 10 ng/ ml of cyclosporine A inhibited the production of 80% IL-22. 3.3 PBMC s Add-in Conclusion: The ability of IL-22 to secrete IL-22 in patients with active BD and normal human rIL-23 was significantly inhibited after the culture of Celecoxib or Cyclosporin A. In active BD patients, the increase in the expression of IL-22 may can be IL-23 induced by the upstream IL-23, suggesting that IL-23 in BD/ IL-2 2. The presence of the pathway. Dexamethasone and cyclosporine A inhibit the production of IL-22. Part III IL-22 Expression of IL-22 in the peripheral blood of patients with VKH and its significance in the analysis of the expression of IL-22 in the peripheral blood of patients with VKH 2. Methods: 2.1 The serum, PBMCs and CD4 ~ + T cells were detected by enzyme-linked immunosorbent assay (ELISA) in the culture of serum, PBMCs and CD4 ~ + T cells. 22. The proportion of IL-22 ~ + CD4 ~ +/ CD8 ~ + T cells, IL-22 ~ + IL-17 ~ + CD4 ~ + T in the peripheral blood of patients with VKH and normal persons was detected by flow cytometry. norm al group" 涓
本文編號(hào)：2510935
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