【作者】 朱红波；

【导师】 周丽；

【作者基本信息】 泰山医学院 ， 内科学， 2009， 硕士

【摘要】 目的研究非霍奇金淋巴瘤(non Hodgkin lymphoma,NHL)患者血清血管内皮生长因子(serum vascular endothelial growth factor,sVEGF)及血清碱性成纤维细胞生长因子(serum basic fibroblast growth factor,sbFGF)水平与临床特征的关系;探讨抗血管生成药物姜黄素对人非霍奇金淋巴瘤Raji细胞增殖及VEGF表达的影响,为临床抗血管新生治疗提供理论依据。方法ELISA法检测不同病期NHL患者sVEGF、sbFGF水平,并收集临床资料,进行统计分析;ELISA法检测姜黄素对Raji细胞上清中VEGF含量变化的影响;不同浓度姜黄素(5umol/L、10umol/L、25umol/L、50umol/L)作用于Raji细胞24h、48h、72h后,WST-1法检测姜黄素对细胞增殖的影响;倒置相差显微镜下观察Raji细胞形态及生长情况;Annexin-V/PI双标流式细胞术检测Raji细胞凋亡率。结果1. NHL患者sVEGF水平(1)NHL初治化疗前36例,其sVEGF浓度为(443.59±203.48)pg/ml;正常对照组19例,其sVEGF浓度为(90.50±21.42)pg/ml,两组间差异有统计学意义(P<0.01)。(2)化疗后完全缓解(complete remission,CR)者17例,其sVEGF浓度为(96.34±38.84)pg/ml;部分缓解(partial remission,PR)者11例,其sVEGF浓度为(109.54±46.37)pg/ml;复发进展(palindromia,PD)者8例,其sVEGF浓度为(553.96±250.08)pg/ml,初治化疗前sVEGF明显高于CR者(P<0.01)、也高于PR者(P<0.05);PD者sVEGF比初治化疗前稍高,但差异无统计学意义(P=0.526>0.05)。(3)第一疗程达CR者9例,其化疗前sVEGF浓度为(152.11±57.98)pg/ml;未达CR者27例,其化疗前sVEGF浓度为(540.75±124.45)pg/ml,差异有统计学意义(P<0.05)。(4)比较36例初治化疗前sVEGF与多个临床相关因素的关系,结果显示Ⅲ～Ⅳ期患者sVEGF水平高于Ⅰ～Ⅱ期者(P<0.05);高中危～高危组sVEGF水平高于低危～低中危组(P<0.05);LDH升高者sVEGF水平明显高于LDH正常者(P<0.05);β2-Mg升高者sVEGF水平明显高于β2-Mg正常者(P<0.05);而sVEGF水平与性别、年龄、全身症状及结外病变数目均无显著相关性(P均>0.05)。2. NHL患者sbFGF水平(1)NHL初治化疗前36例,其sbFGF浓度为(314.47±132.01)pg/ml;正常对照组19例,其sbFGF浓度为(78.55±36.35)pg/ml,两组间差异有统计学意义(P<0.05)。(2)化疗后CR者17例,其sbFGF浓度为(82.28±34.85)pg/ml;PR者11例,其sbFGF浓度为(126.21±61.02)pg/ml;PD者8例,其sbFGF浓度为(338.40±161.12)pg/ml。初治者sbFGF浓度明显高于CR、PR者(P均<0.05);PD者sbFGF浓度比初治化疗前稍高,但差异无统计学意义(P=0.246>0.05)。(3)第一疗程达CR者9例,其化疗前sbFGF浓度为(151.14±35.09)pg/ml;未能达CR者27例,其化疗前sbFGF浓度为(346.39±121.40)pg/ml,差异有统计学意义(P<0.05)。(4)比较36例初治患者sbFGF与多个临床相关因素的关系,结果显示Ⅲ～Ⅳ期患者sbFGF水平高于Ⅰ～Ⅱ期者(P<0.05);高中危～高危组sbFGF水平高于低危～低中危组(P<0.05);LDH升高者sbFGF水平明显高于LDH正常者(P<0.05);β2-Mg升高者sbFGF水平明显高于β2-Mg正常者(P<0.05);而sbFGF水平与性别、年龄、全身症状及结外病变数目均无显著相关性(P均>0.05)。3. sVEGF与sbFGF的相关性pearson相关分析表明sVEGF与sbFGF具有显著正相关性(r=0.548,P<0.05)。4.姜黄素对Raji细胞上清VEGF含量的影响ELISA法检测10、25umol/L姜黄素对Raji细胞作用24h后上清液中VEGF含量的变化,结果显示,对照组上清液中VEGF浓度为(707.92±55.92)pg/ml;而10、25umol/L姜黄素处理组细胞上清液中VEGF浓度分别为(369.73±14.22)pg/ml、(178.24±21.18)pg/ml,与对照组相比两者均显著性降低(P均<0.05)。5.姜黄素处理Raji细胞后WST-1法检测结果5umol/L姜黄素对Raji细胞无明显抑制作用;10μmol/L、25μmol/L、50μmol/L姜黄素对Raji细胞增殖有显著抑制作用,呈浓度和时间依赖性。6.姜黄素处理Raji细胞后形态变化倒置相差显微镜下可见Raji细胞由成团生长变为单个散在,细胞数明显减少,细胞体积变小,折光性减弱,有的细胞膜破裂,可见细胞碎片。7.姜黄素对Raji细胞凋亡的影响流式细胞仪检测浓度为10、25umol/L姜黄素对Raji细胞凋亡的影响,结果示随着姜黄素作用浓度及时间增加,细胞凋亡率随之增加。作用24h,Raji细胞凋亡率分别为11.12%、25.07%;作用48h,凋亡率分别为14.74%、33.50%;作用72h,凋亡率分别为25.11%、55.98%,均高于对照组1.75%、2.25%、2.77%的凋亡率(P均<0.01)。结论1. NHL患者sVEGF、sbFGF水平显著高于正常对照,并与临床分期、IPI预后分组、LDH及β2-Mg有关。提示NHL的发病与血管新生有关,且可联合其他指标一起作为病情监测、预后评估的指标。2.姜黄素能够抑制Raji细胞分泌VEGF,其抑制作用存在浓度依赖性。提示姜黄素具有抑制血管新生的作用,通过其作用达到抗NHL的目的,且为其他抗血管新生药物应用于NHL治疗提供参考。3.姜黄素在一定浓度范围内可以明显抑制Raji细胞增殖,诱导Raji细胞凋亡,并呈浓度和时间依赖性。说明抑制血管新生有可能达到诱导Raji细胞凋亡的作用。更多还原

【Abstract】 ObjectiveThis study focused on the expression level of serum vascular endothelial growth factor(sVEGF) and serum basic fibroblast growth factor(sbFGF) in non Hodgkin lymphoma(NHL) patients and the relationship between them and clinical features; approach the affects of curcumin on proliferation and VEGFs′expression in Raji cells. The study provides a theory evidence for therapy of anti-vessel neogenesis in clinic.MethodsWe measured the sVEGF and sbFGF in NHL patients by ELISA, and collect clinical materials and analyze the data with SPSS. After Raji cells were treated with curcumin at different concentrations(5umol/L,10umol/L,25umol/L,50umol/L) and different time (24h, 48h,72h), cell proliferation was tested by WST-1 assay; the morphological changes of cells were observed by inverted phase contrast microscope; the apoptosis of Raji cells was studied by Annexin V/PI double-labeled flow cytometry; the change of VEGF contents in cultured supernatants of Raji cells treated with different concentratins of curcumin were measured by ELISA.Results1. The sVEGF levels in NHL patients(1) The sVEGF levels in untreated 36 NHL patients is 443.59±203.48pg/ml, significantly higher than that of healthy controls(90.50±21.42pg/ml)(P<0.01).(2) The sVEGF levels in 17 complete remission(CR) patients is 96.34±38.84pg/ml, 11 partial remission(PR) patients is 109.54±46.37pg/ml, and 8 palindromia(PD) patients is 553.96±250.08pg/ml. The sVEGF levels of untreated NHL patients was significantly higher than that of patients in CR (P<0.01), and was also higher than that of patients in PR(P<0.05); the sVEGF levels with PD was significantly higher than that of healthy controls(P<0.01), and was also higher than that of untreated patients, but no significance between them(P=0.526).(3) The sVEGF levels in 9 CR patients in the first cycle chemotherapy is 152.11±57.98 pg/ml, significantly lower than the sVEGF levels in 27 not CR patients in the first cycle chemotherapy (540.75±124.45 pg/ml)( P<0.05).(4) The sVEGF levels in NHL patients with advanced Ann Arbor stage(P<0.05)、high IPI scores(P<0.05) were significantly higher. Compared with those with normal LDH levels, sVEGF levels in NHL patients with higher LDH were elevated(P<0.05); Compared with those with normalβ2-Mg levels, sVEGF levels inβ2-Mg patients with higherβ2-Mg were elevated(P<0.05). But there were no difference about sVEGF levels in untreated NHL patients with different gender, age, B symptoms and number of extranodal site involved(P>0.05).2. The sbFGF levels in NHL patients(1) The sbFGF levels in untreated 36 NHL patients is 314.47±132.01pg/ml, significantly higher than that of healthy controls(78.55±36.35pg/ml) (P<0.05).(2) The sbFGF levels in 17 CR patients is 82.28±34.85pg/ml, in 11 PR patients is 126.21±61.02pg/ml, and in 8 PD patients is 338.40±161.12pg/ml. The sbFGF levels of untreated NHL patients was significantly higher than that of patients in CR and in PR(P<0.05); the sbFGF levels with PD was significantly higher than that of healthy controls(P<0.01), and was also higher than that of untreated patients, but no significance between them(P=0.246).(3) The sbFGF levels in 9 CR patients in the first cycle chemotherapy is 151.14±35.09 pg/ml, significantly lower than the sbFGF levels in 27 not CR patients in the first cycle chemotherapy (346.39±121.40pg/ml)( P<0.05).(4) The sbFGF levels in NHL patients with advanced Ann Arbor stage(P<0.05)、high IPI scores(P<0.05) were significantly higher. Compared with those with normal LDH levels, sbFGF levels in NHL patients with higher LDH were elevated(P<0.05); Compared with those with normalβ2-Mg levels, sbFGF levels inβ2-Mg patients with higherβ2-Mg were elevated(P<0.05). But there were no difference about sbFGF levels in untreated NHL patients with different gender, age, B symptoms and number of extranodal site involved(P>0.05).3. The correlation between sVEGF and sbFGFThere is positive correlation between sVEGF level and sbFGF level in NHL patients(r=0.548,P<0.05).4. The change of VEGF contents in cultured supernatants of Raji cells The change of VEGF contents in cultured supernatants of Raji cells treated with different concentratins of curcumin(10μmol/L, 25μmol/L) were measured by ELISA, the levels of VEGF were 369.73±14.22pg/ml, 178.24±21.18pg/ml, and significantly lower than that of the control group′s (707.92±55.92pg/ml).5. The results of WST-1There was no obviously refrain proliferation of Raji cells in 5umol/L curcumin; but in certain range of concentration (10μmol/L, 25μmol/L, 50μmol/L ), curcumin could obviously refrain proliferation of Raji cells in a dose and time dependent manner.6. The morphological changes of Raji cellsThe morphological changes of cells were observed by inverted phase contrast microscope, we can found that the Raji cells turned into disorder from conglobation, the cell population obviously was decreased and the cell volume shrinked, the refraction weakened, the cell membrance disrupted, and we can see cell debris.7. The apoptosis of Raji cellsThrough flow cytometry, we can found that the apoptosis rate of Raji cells was increased as the concentration of curcumin and action time were increased.Conclusions1. Serum VEGF、bFGF levels of NHL patients were higher than that of healthy adults, and were associated with advanced Ann Arbor stage, high IPI scores, LDH, andβ2-Mg. Angiogenesis perhaps involved in the development of NHL, and might be used to monitor patients conditions、evaluate prognosis with other indexes.2. Curcumin can inhibit VEGF secretion of Raji cells , its relied on concentrations. These results suggest that curcumin can anti-vessel neogenesis.3. Curcumin can significantly inhibit Raji cells proliferation in certain range of concentration, its relied on concentrations and times.更多还原