| | | 柔红霉素（DNR）荧光直方图诊断白血病细胞耐药的意义
| | 医学毕业论文【摘要】目的医学毕业论文：建立检测细胞内柔红霉素（DNR）的医学毕业论文荧光直方图诊断白血病细胞耐药的方法。材料与方法：选取15例临床耐药的急性髓性白血病（AML），5初治AML进行研究，用流式细胞仪（FCM）检测细胞内DNR的荧光强度，根据其强弱程度划分成耐药区和药敏区，其直方图主峰左移（LSMP）诊断为耐药；直方图右移（RSMP）诊断为药敏。耐药指数（DRI），即R/S（R为耐药细胞数，S为药敏细胞数）≥2为耐药，＜2为药敏。荧光指数（FI）=平均荧光强度×DRI，FI≥200为耐药，FI＜200为药敏。结果：初治5例AML中，4例为RSMP，DRI=0.23～1.13，FI=50～160，诊断为药敏；另一例为LSMP，RI=3.60，FI=367，诊断为原发耐药。15例临床耐药的AML中，12例为LSMP，DRI=2.59～17.72，FI=265～1041，诊断为经典耐药；其余3例为RSMP，DRI=0.39～0.67，FI=57～83，诊断为再生耐药。结论：用FCM检测白血病细胞内DNR的荧光直方图，结合DRI、FI以及临床耐药表现和耐药细胞荧光强度的诊断界点的方法，我们可以快速、简便、直观地在临床上及时发现和评价AML耐药的类型和程度，可为制定个体化疗的方案提供依据，也为观察逆转耐药的疗效提供了一个方法。
Significance of fluorescent histogram of daunorubicin(DNR) in
diagnosis of drug resistance to leukemic cells
【Abstract】Objective：To develop a fluorescent histogram of intracellular daunorubicin(DNR) as a diagnostic method for drug resistance in acute myeloid leukemia(AML).Methods：15 patients with clinically diagnosed drug resistant AML and 5 patients with previously untreated AML were studied. The intensity of fluorescence of intracellular DNR and the fluorescent histogram were determined by flow cytometer(FCM).According to a degree of the intensity, both drug－resistan t and－sensitive zones were marked off. Left shift of main peak(LSMP) with the fluoresent histogram may be diagnosed as drug resistance, and right shift of main peak(RSMP) as drug sensitivity. R/S stood for drug resistant index(DRI);R for the number of drug resistant cells, and S for drug－sensitive cells. DRI≥ 2 was drug－resistant or DRI ＜2 was drug-sensitive. Fluorescent index(FI) was the product of mean fluorescent intensity (MFI) multiplied by DRI. FI≥ 200 meant drug-resistant, while FI＜ 200 meant drug-sensitive.Results：Among 5 patients with previously untreated AML, 4 patients were diagnosed as drug-sensitive for RSMP, DRI=0.23～ 1.13,FI=50～ 160,and one patient was diagnosed as primary drug-resistant for LSMP,DRI=3.60, FI=367. In 15 patients with clinically drug resistant AML,12 patients were diagnosed as classical resistance for LSMP,DRI=2.59～ 17.72,and FI=265～ 1041,and 3 patients as regrowth drug resistance for RSMP,DRI=0.39～ 0.67,and FI=57～ 83.Conclusion：Clinically, it was a rapid, convenient and visual way, which by FCM mapping the fluorescent histogram of intracellular DNR, in combination with ;DRI, FI, a clinically drug-resistant manifestation and a cut off point for diagnosis of the fluorescent intensity of drug-resistant cells, we may in good time find and value a type and a degree of drug resistance to AML. Moreover ,it may provide a basis for working out an individually therapeutic scheme and a method for an observation in drug-resistant reversal treatment as well.
Key words：Daunorubicin(DNR); Fluorescent histogram; Drug resistance; Flow cytometer (FCM); Leukemia
Drug resistance in leukemia was a leading cause underlying chemotherapeutic failure. Its degree and its type were close associated with the chemotherapeutic scheme for each patient. Still, an urgent problem to solve in clinic is how to find and evaluate the resistance quickly, conveniently and timely. Daunorubicin (DNR) produces red fluorescence with laser excitation at 488 nm wavelength. Utilizing it, drug resistance was detected through measuring DNR fluorescent intensity in the cells. It was only in a few minutes that flow cytometer(FCM) could analyse it for thousands of cells and draw into a fluorescent histogram that represented the marks of drug resistance to leukemic cells. Hence, we tried to investigate the diagonstic significance for the fluorescent histogram in classical and in regrowth drug resistance in leukemia.
1 MATERIALS AND METHODS
1.1 Patients
15 patients with acute myelogenous leukemia(AML),who manifested drug resistance clinically according to a clinically drug－resistant standard〔1〕,were eligible for this study.9 males,6 females, age 17～ 52 years old. 5 patients with previously untreated AML were investigated as control.3 males,2 females, age 32～ 48 years old. All of them were diagnosed with a clinical, cell morphology and cell chemistry method in our hospital. Mononuclear cells were obtained with a standard way, i.e. Ficoll Hypaque density gradient separation, in which the number of leukemic cells were more than 90％.
1.2 Reagents
DNR(Farmitalia Carlo Erba,Italy) and RPMI 1640(Gibco－BRL,USA).
1.3 Assay of DNR fluorescent intensity in leukemic cells
Leukemic cells were washed twice with RPMI 1640 which contained 10％ calf serum. The concentration of cells was adjusted&nbs p;to 1× 106 /ml.1 μg/ml DNR was added and incubated for 1 hour at 37℃ ,5％ CO2.Then dissociated DNR was washed twice with RPMI 1640 at 4℃. 1 ml RPMI 1640 at 4℃ was added, the fluorescent intensity of cells was detected by FCM ( Becton Dickinson, USA ). 488nm wavelength was used. FCM was first adjusted with fluorescent balls , and CV value was lower than 3％.10 000 cells were assayed for each sample. Sample without DNR was regarded as control. The detected results were drawn into the histogram with software CELLQuest according to fluorescent intensity that represented intracellular DNR concentration. The ordinate was cell number and the abscissa was logarithmic fluorescent intensity.
1.4 Data analysis
According to other article〔2〕,the cut-off point of DNR fluorescent intensity in drug－resistant cells was 100. Passing through this point, fluorescent intensity was divided into two zones, namely, ＜100 was the zone of drug resistance, and ≥100 was the zone of drug sensitivity. The left shift of main peak(LSMP) in DNR fluorescent histogram meant that it located in the drug－resistan t zone. The previously untreated patient was diagnosed as primary drug resistance. If the patient manifested drug resistance clinically, he was diagnosed as classical drug resistance. The right shift of main peak （RSMP）meant that it located in the drug－sensitive zone. If the patient manifested drug resistance clinically, he was diagnosed as regrowth drug resistance. R stood for cell number of drug resistance;S stood for cell number of drug sensitivity. The drug resistant index(DRI)=R/S. If it≥ 2,it meant drug resistant, and＜2 meant drug sensitive. Fluorescent index (FI)=mean fluorescent intensity(MFI)× DRI. FI＜200 indicated sensitivity, and≥200 indicated resistance; the larger the value of them, the higher the degree of drug resistance.
2 RESULTS
The DNR fluorescent histogram. 4 cases of previously untreated AML displayed RSMP in the fluorescent histogrom, in which the main peak were high－narrowed(Fig.1), DRI=0.23～1.13,and FI=50～160.These patients were diagnosed as drug sensitive. One case of previously untreated AML displayed LSMP, in which the main peak was high－narrowed, too (Fig.2),DRI=3.60,and FI=367. The patient was diagnosed as primary drug resistant.15 cases of AML showed drug resistance clinically, in which 12 cases displayed LSMP(Fig.3), including in 3 cases represented a subpeak near the main peak(Fig.4), DRI=2.59～17.72, and FI=265～1041.These patients were diagnosed as classical drug resistance. 3 cases of AML who showed drug resistant clinically displyed RSMP, in which the shape of the main peak was between high－narrowed and low－broadened peaks, DRI=0.39～ 0.67,and FI=57～ 83. They were diagnosed as regrowth drug resistance(Fig.5).
3 DISCUSSION
The mechanism of drug resistance to leukemic cells was very complex,one of which, chemotherapeutic agents may be pumped out of these cells. In some confirmed experiments, in vitro measurements of DNR concentration functionally disclosed in vivo drug resistance to cells. FCM could determine the DNR fluorescent intensity which represented the variations of the DNR concentration in cells. As some experiments had been demonstrated, there was an inverse proportion of the intensity to the concentration〔2,3,4,5〕.
At present, MFI often means drug resistant to a cell population, and&nb sp;it depends on two factors, i.e. the fluorescent intensity and the cell number which generate fluorescence. DRI could account for the degree of drug resistance. The larger the value of it, the higher the degree. Meanwhile, referred to Webb’s method〔6〕，FI accounted for the degree of drug resistance. For example, firstly, a patient in this study was diagnosed as drug sensitive for MFI=121.04 but secondly, he was diagnosed as drug resistant after a combination of DRI=2.60 and FI=314 was considered. Con－sequently, the second diagnosis was consistant with his manifestation of clinical drug resistance.
Leukemic cells in vivo belong to a heterogenous population with different degrees of drug resistance. From the fluorescent histogram, definite number of resistant cells were presented in all the patients, previously untreated or clinically drug－resistant, whose number will make a characteristics or a phenotype of drug resistance. Besides the main peak, one or two subpeaks were sometimes seen in the drug resistant zone. This explained the presence or development of resistant subpopulations, and also explained the heterogenicity& nbsp;of drug－resistant cells.Because they could survive the chemotherapeutic agents, there was some prognostic significance in observing the variations of their number underlying relapse and drug resistance in leukemia. Additionally, the alteration of their degree and their type of drug resistance, to some extent, would ensue from the growing of cell colonies in leukemia.
From what we had observed, there were two main peaks in the histogram：one was a high－narrowed peak that meant the resistant or sensitive cells were concentrated in a certain degree; the other was a low broadened peak that meant the cells were dispersive. The former, including drug－resistant andsensitive cells which centered on a certain phase, may have a good effect on agents. The latter, which cells had different degree of drug resistance, may have a poor effect on agents. Furthermore, the more left－shifting of the main peak, the higher the degree of drug resistance. As for secondary drug resistance, the main peak, which its shape varied from high－narrowed to low－broadened and its position varied from right to left, may suggest that the drug sensitive cells have changed into the drug－resistant cells.
FCM can in a few minutes determine the DNR fluorescent parameters of ten thonsands cells and draw the fluorescent histogram and it takes four hours or so to reach the analytic conclusion. So, it provides a rapid, convenient and visual basis for designing individually therapeutic regimens and provides a method for a clinical observation in reversing drugresistant treatment as well.
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