本文關鍵詞：流式細胞術檢測Cyclin D1和BCL-2在B細胞淋巴瘤診斷中的意義，由筆耕文化傳播整理發(fā)布。

        目的：探索流式細胞術檢測Cyclin D1和BCL-2在B細胞淋巴瘤診斷中的方法、可行性及臨床意義。方法:1）收集我院血液內科收治確診的血液病患者45例,其中男22例，女23例，年齡8-84歲，其中HL3例，CLL15例，DLBCL11例，MCL3例，MZL2例，F(xiàn)L2例，NK-T細胞淋巴瘤4例，，T細胞淋巴瘤5例，收治后詳細記錄治療經(jīng)過，并進行后期隨訪和療效評定。根據(jù)2008年WHO淋巴瘤分類標準，將淋巴瘤患者分類。根據(jù)張之南《血液病診斷與療效指南》療效判定，將接受治療的患者分為完全緩解（CR），不確定的完全緩解（CRu），部分緩解（PR），疾病穩(wěn)定（SD），復發(fā)（適用于取得CR/Cru者）和疾病進展（PD）。2）按照年齡、性別相匹配的原則，選取在我院接受常規(guī)體檢且各項檢驗指標均在正常范圍內的健康體檢者25名作為健康對照組。3）選取病理科已確診的Cyclin D1或BCL-2陽性病例組為陽性對照組（8例），病理已確診的Cyclin D1或BCL-2陰性的病例組為陰性對照組（7例）。采用四色直接免疫熒光素法標記細胞抗原，包括細胞膜表面和胞核抗原，如：CD5、CD19、CD20、CD23、CD10、 Cyclin D1、BCL-2等，以CD19+細胞設門，多參數(shù)流式細胞術檢測各亞型淋巴瘤患者外周血、骨髓或淋巴結Cyclin D1及BCL-2表達水平。所有數(shù)據(jù)用SPSS13.0統(tǒng)計軟件處理分析；各計量資料采用均數(shù)±標準差((?)±s)表示，兩組數(shù)值符合正態(tài)分布及方差齊性采用t檢驗，否則采用非參數(shù)檢驗；多組之間的比較，數(shù)值符合正態(tài)分布和方差齊性采用方差分析，否則采用非參數(shù)檢驗，P<0.05為差異具有統(tǒng)計學意義。比較兩種檢測方法的差異采用配對卡方檢驗，P<0.05為差異具有統(tǒng)計學意義。結果1)本課題對25例正常成人志愿者Cyclin D1MFI及BCL-2MFI進行了分析后得出x±s值及95％可信區(qū)間，從而得出流式檢測正常參考值范圍，并由此得出判斷Cyclin D1的陽性閾值為Cyclin D1MFI＞0.5；同樣，判斷BCL-2的陽性閾值為BCL-2MFI＞2.7。2)根據(jù)FCM確定的Cyclin D1及BCL-2陰陽性診斷標準，單純與病理免疫組化結果進行對比發(fā)現(xiàn)，F(xiàn)CM檢測MCL患者的Cyclin D1靈敏度及特異性為100％，檢測FL患者的BCL-2靈敏度及特異性也為100％，且FCM檢測Cyclin D1及BCL-2結合免疫表型等對病理不能分類淋巴瘤明確了亞型5例。3)比較健康對照組與全體B細胞淋巴瘤患者組外周血Cyclin D1和BCL-2MFI顯示：B細胞淋巴瘤患者Cyclin D1(1.824±0.312)和BCL-2(4.259±0.541)的表達水平較正常對照組(分別為0.356±0.159,1.938±0.324)明顯升高，兩者均具有統(tǒng)計學顯著性差異（p＜0.0001），4)不同亞型淋巴瘤患者外周血Cyclin D1MFI的表達有差異，霍奇金淋巴瘤患者Cyclin D1MFI（0.386±0.112）明顯低于非霍奇金淋巴瘤患者（1.623±1.987）（p＜0.001），而在非霍奇金淋巴瘤患者中，套細胞淋巴瘤患者CyclinD1陽性最高（100％），其余亞型均為陰性；不同亞型淋巴瘤患者外周血BCL-2的表達情況有差異，霍奇金淋巴瘤患者BCL-2(2.045±0.877)明顯低于非霍奇金淋巴瘤(4.045±0.499)患者（p＜0.001），在非霍奇金淋巴瘤中，T細胞淋巴瘤均為陰性，F(xiàn)L、MCL表達陽性率最高（100％），其次為MZL（50％）、CLL（30％）、DLBCL(45％)。5)在Cyclin D1或BCL-2陽性的淋巴瘤患者中，對治療后臨床判斷達部分緩解以上的患者進行流式細胞儀檢測，發(fā)現(xiàn)Cyclin D1（治療前3.099±0.349；治療后1.008±0.279）或BCL-2（治療前7.814±1.030；治療后3.131±0.522）的平均熒光強度均有顯著降低（p＜0.001）。結論：1）流式細胞術檢測淋巴細胞中Cyclin D1及BCL-2的表達方法簡便可行；2）建立流式細胞術檢測淋巴細胞中Cyclin D1及BCL-2的參考值；3）FCM檢測CyclinD1及BCL-2對B細胞淋巴瘤的診斷分型有意義，具有很好的靈敏度和特異性；4）淋巴瘤患者治療后Cyclin D1或BCL-2表達下調，其表達變化與治療效果有關，可以作為臨床觀察治療效果的指標之一。

    Objective： To explore the method of detecting Cyclin D1and BCL-2by flowcytometry, as well as feasibility and significance.Methods:1） Patients with hematologic diseases in final diagnosis werecollected and recorded with45cases in detail,22were male,23female,age8-84, including HL3cases, CLL15cases, DLBCL11cases, MCL3cases,MZL2cases,FL2cases， NK-T cell lymphoma4cases, T cell lymphoma5cases,including treatment, follow-up and evaluation of curative effect.According to the2008WHO lymphoma classification criteria, patients wereclassified. According to Zhang Zhinan 《blood disease diagnosis andtreatment guide》 to judge the curative effect, treated patients weredivided into complete remission (CR), does not determine the completeremission (CRu), partial remission (PR), stable disease (SD),recurrence (for CR/Cru) and disease progression (PD).2）Accordingto the age and gender matching principle, selected routine physicalexamination and all tests were within the normal range in20healthy personsas a healthy control group.3）Select the pathology confirmed Cyclin D1or BCL-2positive cases as positive control group (8cases), select thepathology confirmed Cyclin D1or BCL-2negative cases as negative controlgroup (7cases). Four color monoclonal antibodies directly labeled withimmunofluorescence were used to analyze the surface and cytoplasma antigens,such as CD5, CD19, CD23, CD20, Cyclin D1, BCL-2and so on, CD19+cells wereset as door, we used multi-parameter flow cytometry to investigate peripheral blood Cyclin D1and BCL-2subsets in lymphoma patients.All datawas analyzed by statistical software SPSS13.0; the measurement data wasexpressed as mean±standard deviation ((?)±s). We used t test when twosets of values in line with normal distribution and homogeneity of variance,or using non-parametric test; we used analysis of variance when multiplesets of comparisons between values in line with normal distribution andhomogeneity of variance, or we used non-parametric tests,P<0.05meant thedifference was statistically significant. We used paired chi-square testto compare differences in detection methods, P<0.05meant the differencewas statistically significant.Results：1）, The subject of the25normal adult volunteers Cyclin D1MFIwere analysed x±s values and95%confidence interval, so that the CyclinD1MFI>0.5is positive, thus establishing the Cyclin D1diagnosticcriterion; BCL-2MFI were also analysised x±s values and95%confidenceinterval in25cases of normal adult volunteers, so that the BCL-2MFI>2.7is positive, thus establishing the BCL-2diagnostic criterion;2）,Cyclin D1and BCL-2diagnosed by FCM criteria of positive and negative,simplely compared with immunohistochemistry results of pathological in MCLpatients, FCM analysis of Cyclin D1sensitivity and specificity of100%,the detection of patients with FL BCL-2sensitivity and the specificityis100%.FCM detection of Cyclin D1and BCL-2with the combination of immunephenotype clear5cases of pathology can not be classified by lymphomasubtype.3) Compared with the healthy control group, all the B-cell lymphomain patients with peripheral blood of Cyclin D1(1.824±0.312) and BCL-2MFI (4.259±0.541) was significantly much more increased expressionlevels than normal control group (0.356±0.159,1.938±0.324), bothstatistically significant difference (p <0.0001).4) Different subtypes of lymphoma in peripheral blood of patients express differences of CyclinD1MFI.In patients with Hodgkin’s lymphoma of Cyclin D1MFI (0.386±0.112)was significantly lower than non-Hodgkin’s lymphoma patients (1.623±1.987)(p <0.001).In non-Hodgkin’s lymphoma patients, mantle cell lymphomapatients with Cyclin D1-positive (100%), and the remaining subtypes werenegative; different subtypes of lymphoma in peripheral blood of patientswith BCL-2expression differences, in patients with Hodgkin’s lymphomaBCL-2(2.045±0.877) was significantly lower than non-Hodgkin’s lymphoma(4.045±0.499) patients (p <0.001), in non-Hodgkin’s lymphoma, T celllymphoma express lowest, positive rate of MCL and FL was the highest (100%),followed by MZL (50%), CLL (30%), DLBCL (45%).5) lymphoma patients withCyclin D1or BCL-2-positive, more than patients detected the clinicaljudgment of partial remission after treatment by flow cytometry and foundthat Cyclin D1(before treatment3.099±0.349; after treatment,1.008±0.279)or expression of BCL-2(before treatment7.814±1.030; aftertreatment,3.131±0.522) mean fluorescence intensity are significantlylower (p <0.001).Conclusion:1, the method of flow cytometry for detection of Cyclin D1andBCL-2is feasible;2, Establishment of Cyclin D1and BCL-2reference valuein flow cytometry;3, FCM analysis of cyclin D1and BCL-2Diagnosticclassification of B-cell lymphoma meaningful, with good sensitivity andspecificity.4. Cyclin D1or BCL-2of Lymphoma patients after treatmentdownregulation, and its expression change with treatment, can be used asa clinical observation on evaluate the treatment.

        流式細胞術檢測Cyclin D1和BCL-2在B細胞淋巴瘤診斷中的意義

縮略詞表5-6摘要6-8Abstract8-10引言11-13材料方法13-18統(tǒng)計學方法18-19結果19-28討論28-34結論34-35參考文獻35-38附錄38-40致謝40-41綜述41-49    參考文獻46-49

  本文關鍵詞：流式細胞術檢測Cyclin D1和BCL-2在B細胞淋巴瘤診斷中的意義，由筆耕文化傳播整理發(fā)布。