摘要
乳腺癌是妇女中最常见的恶性肿瘤,目前的治疗方法包括手术、放疗、化疗、内分泌治疗及生物治疗,但效果仍不理想。肿瘤干细胞(Cancer Stem Cell,CSC)学说的提出为彻底根除癌细胞,治愈乳腺癌带来了希望。CSC是一群具有自我更新能力和不定向分化潜能的细胞,是肿瘤不断生长的根源,也是肿瘤发生、扩散、复发等过程中的“起始细胞”和“动力细胞”。乳腺癌中的CSC不仅对彻底治疗乳腺癌具有重要意义,与乳腺癌患者的预后也密切相关。本研究首先探讨了乳腺癌CSC新的标志物ALDH1和CD55表达在乳腺癌预后评估中的应用价值,然后应用微球体培养法和化疗法分别对小鼠乳腺癌细胞系4T1中的CSC在体外和小鼠体内进行了富集,得到了富含乳腺癌CSC的细胞群和富含乳腺癌CSC的小鼠模型,最后应用影响CSC微环境的药物小白菊内酯(Parthenolide,PTL)在体外和小鼠体内对CSC进行杀伤实验,为临床应用PTL治疗乳腺癌提供依据。本研究共分为三个部分:
第一部分ALDH1和CD55表达在乳腺癌预后评估中的意义
目的:探讨乳腺癌CSC新的标志物ALDH1或CD55表达与乳腺癌组织学类型、分级等疾病特征的关系,明确ALDH1和CD55表达在乳腺癌预后评估中的应用价值。
方法:应用免疫组化法对65例随访5~8年的乳腺癌患者组织标本进行ALDH1和CD55表达检测。ALDH1表达于胞质,阳性表达细胞呈棕黄色,阴性表达细胞呈蓝紫色。选取1张切片中的3个代表性视野,均有ALDH1阳性表达细胞者判为ALDH1阳性标本。CD55表达于胞质和胞膜,强阳性表达细胞呈棕黄色,弱阳性表达细胞呈浅黄色,阴性表达细胞呈蓝紫色。CD55高表达标本为CD55强阳性表达细胞≥5%。据以上标准判定ALDH1阳性表达及CD55高表达标本,分析乳腺癌患者各临床疾病特征与CD55高表达及ALDH1阳性表达的关系。分析患者5年生存率与CD55高表达、ALDH1表达及其它临床疾病特征的相关性,利用Cox比例分析模型进行乳腺癌患者预后的单因素分析,进而行多因素非条件Logistic回归分析进一步筛选影响患者预后的独立危险因素。
结果:
1正常乳腺组织ALDH1表达为阴性或极弱阳性,乳腺癌组织标本中ALDH1的阳性表达率为24.6%(16/65);正常乳腺组织CD55为阴性或低表达。在乳腺癌组织标本中,高表达CD55的标本占33.8%(22/65)。
2 ALDH1阳性表达和CD55高表达与乳腺癌患者绝经状况、肿瘤组织学类型、组织学分级、肿瘤直径、是否发生淋巴结转移、雌激素受体和孕酮受体表达情况、表皮生长因子受体2(her2)表达情况、术后是否接受其他治疗均无关(P>0.05);与患者治疗后的肿瘤复发密切相关(P<0.01)。
3 ALDH1阳性、CD55高表达患者的5年生存率与ALDH1阴性、CD55低表达患者相比显著降低(P<0.001)。
4 ALDH1表达、CD55高表达、肿瘤直径、组织学分级、her2表达阴性、术后未接受治疗6项因素与乳腺癌患者5年生存率有关,是影响乳腺癌患者预后的独立影响因素。
结论:ALDH1阳性表达和CD55高表达与肿瘤复发密切相关,是影响乳腺癌患者预后的独立因素,但与乳腺癌患者其它疾病特征无关。
第二部分小鼠乳腺癌细胞系4T1中CSC的体外富集及富含乳腺肿瘤CSC的动物模型制备
目的:应用微球体培养法和化疗法分别对小鼠乳腺癌细胞系4T1的CSC在体外和小鼠体内进行富集,得到富含乳腺癌CSC的细胞群和富含乳腺癌CSC的小鼠模型,并对其特征进行研究,为进一步研究如何杀伤乳腺癌CSC奠定基础。
方法:
1 CSC微球体培养法:4T1细胞以2×104/mL接种于无血清培养基(SFM)中,静止培养3天,离心换液,当培养瓶中形成由100~200个细胞构成的细胞球后,用稀释的胰蛋白酶-EDTA消化传代,约6~7天传代一次。
2富含乳腺癌CSC的荷瘤小鼠模型制备:4T1细胞悬液0.2ml(含有1.5×106个细胞)接种于小鼠右肩胛皮下,观察瘤块形成,直径达0.1cm左右时,给予小鼠0.2mg/ml的5-FU0.2ml,给药方式为腹腔注射,用药时间为每天一次,共用七天,七天后改为一周一次。四周后脱颈处死小鼠,无菌取小鼠肿瘤,以组织研磨器研磨制成细胞悬液,用PBS调整活细胞浓度为7.5×106/ml。重复以上操作制作第二代富集CSC的小鼠模型,共制作四代小鼠模型。
3 CSC的检测:应用流式细胞仪检测CD44+CD24-/low细胞的含量,应用HOECHST33342染色法观察计数无荧光染色侧群(SP)细胞比例,应用荧光定量PCR法检测细胞耐药基因MDR1、BCRP、ALDH1 mRNA表达。应用免疫组化法检测肿瘤组织CD55和ALDH1的表达,无血清悬浮培养观察肿瘤组织细胞微球体形成效率。致瘤性检验验证富集的CSC成瘤性。
结果:
1 4T1细胞在SFM中可形成能够连续传代的细胞球,第10代微球体细胞中CD44+CD24-/low细胞含量可达68.9±3.78%,侧群(SP)细胞比例为56.1±5.18%,MDR1、BCRP和ALDH1 mRNA表达与SSM培养4T1细胞对照相比分别上调了1.63±0.21倍、2.14±0.21倍和3.41±0.53倍。致瘤性检验表明,微球体细胞2×103个细胞即可在小鼠体内成瘤,而SSM培养的4T1细胞至少需要2×105个细胞才可成瘤。
2利用化疗药5-Fu建立了富含乳腺癌CSC的小鼠模型。第三代小鼠模型肿瘤组织CD44+CD24-/low细胞比例可达69.0±1.6%,SP细胞比例为61.3±2.6%,与对照的肿瘤组织相比MDR1、BCRP和ALDH1 mRNA水平分别上调了4.35±0.21倍、6.14±0.47倍和3.78±0.32倍(P<0.01)。ALDH1表达为阳性,CD55强阳性表达细胞数为17.3±1.9%,明显强于对照肿瘤组织。致瘤性检验表明,接种模型组肿瘤组织2×103个细胞即可在小鼠体内成瘤,而对照肿瘤组织至少需要2×105个细胞才可成瘤。
结论:利用无血清微球体培养在体外富集了乳腺癌细胞系4T1中假定的CSC,利用化疗药物制备了富含乳腺癌CSC的小鼠模型,通过多项指标检测及致瘤性检验证实,本实验富集CSC制备富含乳腺癌CSC的小鼠模型是成功的,为进一步研究靶向杀伤CSC奠定了基础。
第三部分小白菊内酯杀伤小鼠乳腺癌肿瘤干细胞的研究
目的:探讨小白菊内酯(Parthenolide,PTL)在体内和体外对乳腺癌CSC增殖及分化的影响,为临床应用PTL治疗乳腺癌提供依据。
方法:
1 PTL杀伤4T1细胞系中CSC的体外实验:无血清悬浮培养的第10代微球体细胞以SFM培养基培养,接种于25ml培养瓶(5×104/mL),25瓶细胞随机分为对照组、5-FU组、PTL组、5-FU+PTL组、PTL+NAC(N-乙酰半胱氨酸)组5组。对照组加入生理盐水20μl,5-FU组加入5-FU(0.1mg/ml) 20μl,PTL组加入PTL(1mg/ml)20μl,5-FU+PTL组加入5-FU(0.2mg/ml) 10μl和PTL(2mg/ml)10μl,PTL+NAC组加入PTL(2mg/ml)10μll和NAC(20mg/ml) 10μl。培养48小时后离心换液用SFM继续培养。7天后观察细胞的成球情况,收集细胞进行检测。
2 PTL杀伤4T1细胞系中CSC的动物实验:雌性BALB/c小鼠共25只,抽签法随机分为对照组、5-FU组、PTL组、5-FU+PTL组、PTL+NAC组5组。无菌手术取经化疗法形成的第三代小鼠移植瘤,将肿瘤组织研磨制成细胞悬液,吸取0.2ml细胞悬液(含1.5×106个细胞)接种于小鼠皮下。接种24小时后对照组小鼠给予生理盐水0.4ml,分别为腹腔0.2ml,肩胛部肿瘤细胞接种部位0.2ml;5-FU组小鼠给予0.2mg/ml的5-FU0.4ml,PTL组小鼠给予1mg/ml的PTL0.4ml, 5-FU+PTL组小鼠给予含0.2mg/ml 5-FU和1mg/ml PTL的混合药物0.4ml,PTL+NAC组给予含1mg/ml PTL和10mg/ml NAC的混合药物0.4ml,给药部位和剂量与空白对照组相同。用药时间为每天一次,共用七天,七天后改为一周一次。四周后脱颈处死小鼠,取肿瘤进行检测。
3 CSC的检测:应用流式细胞仪检测CD44+CD24-/low细胞的含量,应用HOECHST33342染色观察计数无荧光染色侧群细胞(SP)细胞比例,应用荧光定量PCR法检测细胞耐药基因MDR1、BCRP、ALDH1 mRNA表达。应用免疫组化法检测肿瘤组织CD55和ALDH1的表达。
结果:
1 PTL杀伤4T1细胞系中CSC的体外实验结果:经过5-FU、PTL、5-FU+PTL或PTL+NAC处理后用SFM培养,对照组、5-FU组、PTL+NAC组可形成明显的细胞球,PTL组形成的细胞球很少,仅有散在的细胞球存在,5-FU+PTL组存活细胞极少,细胞因凋亡而裂解。对照组、5-FU组、PTL组、PTL+NAC组形成的细胞球中CD44+CD24-/low细胞含量分别为71.2±2.3%、75.6±3.1%、12.3±1.9%、58.1±2.6%;无荧光染色细胞(SP)细胞百分比分别为56.7±3.4%、62.0±2.7%、8.1±1.1%、51.5±2.3%,PTL组CD44+CD24-/low细胞和SP细胞含量明显低于对照组、5-FU组和PTL+NAC组(P<0.01)。
2 PTL杀伤4T1细胞系中CSC的动物实验结果:经过5-FU、PTL、5-FU+PTL或PTL+NAC治疗后,对照组、5-FU组小鼠形成肿瘤的速度和直径远大于PTL组、5-FU+PTL组、PTL+NAC组(P<0.01)。对照组、5-FU组、PTL组、5-FU+PTL组、PTL+NAC组各组小鼠肿瘤组织中CD44+CD24-/low细胞含量分别为57.3±4.1%、68.7±3.2%、42.5±3.7%、39.1±2.9%、50.1±3.1%,无荧光染色细胞(SP)细胞百分数分别为58.1±4.1%、61.3±2.6%、36.9±3.5%、39.2±1.8%、42.7±2.5%,PTL组、5-FU+PTL组、PTL+NAC组CD44+CD24-/low细胞和SP细胞含量明显低于对照组、5-FU组(P<0.05)。5-FU组、PTL组、5-FU+PTL组、PTL+NAC组与对照组相比,荷瘤小鼠肿瘤组织中MDR1相对基因表达量分别为1.11±0.11、0.92±0.12、1.03±0.15、0.94±0.08,各组间比较无统计学差异(P>0.05);BCRP相对基因表达量分别为1.09±0.16、0.97±0.10、1.13±0.11、0.95±0.13,各组间比较无统计学差异(P>0.05)。ALDH1相对基因表达量分别为1.16±0.12、0.38±0.07、0.42±0.09、0.57±0.04, PTL组、5-FU+PTL组、PTL+NAC组明显低于对照组和5-FU组(P<0.05)。
结论: PTL可以靶向乳腺癌CSC,改变CSC的状态,使培养细胞或肿瘤组织CSC含量明显降低。抗氧化剂NAC体外实验中可以明显拮抗PTL这种作用,但在小鼠体内作用不明显。PTL与其它化疗药物联合应用,可增强其它药物的化疗效果,PTL可作为化疗增敏剂使用。
Breast carcinoma is one of the most serious malignancies among females. The current treatment include surgery, radiotherapy, chemotherapy, endocrine therapy and biological treatment, but these method are not satisfactory. To eradicate breast cancer, eradicate cancer cells from the origin should be most effective way. The theory of cancer stem cell (CSC) gives hope for the complete curing breast cancer. CSC is a group of cells which have self-renewing and non-directional differentiation potential, it is the root of tumor continued growth; it is also the "starting cell" and "motivation cell" for tumorigenesis, tumor proliferation, recurrence, etc. CSC is not only a thoroughly therapy way for breast cancer, but also has closely relation with the prognosis of breast cancer. This study first explores the values of the breast cancer CSC new markers ALDH1 and CD55 expression in breast cancer prognosis, then use the serum-free suspension mammosphere culture and chemotherapy way to enrich breast cancer CSC from 4T1 cell lines in vitro and in vivo respectively. The cell populations of CSC and mouse models of CSC were getted. At last we use Parthenolide (PTL) to targeting CSC in vitro and in vivo; it will provide the foundation for the treatment of breast cancer with PTL.
This research is divided into three parts.
Part 1 Prognostic significance of ALDH1 and CD55 expression in breast cancer
Objective: To evaluate the relationship of new breast cancer CSC marker CD55 or ALDH1 expression in breast cancer with the diseases characteristics such as histological type, grading, etc. Identify the prognosis value of ALDH1 and CD55 for breast cancer.
Methods: ALDH1 and CD55 expression in tumor tissue of 65 breast cancer patients which followed up 5 to 8 years was detected with immunohistochemical staining. ALDH1 expressed in the cytoplasm, ALDH1 positive cells showed brown-yellow, negative cells were blue-purple. Select three horizons of a representative slice, all horizons which have ALDH1 positive cells were sentenced as ALDH1 positive specimens. CD55 expressed in the cytoplasm and membrane, strong positive expression cells were brown, weakly positive expression cells were weak yellow. CD55 high expression samples were strong positive cells≥5%. According to the above criteria, determine ALDH1 expression and CD55 high expression samples, analysis the relationship of the clinical disease characteristics with CD55 high expression and ALDH1 expression. Calculated 5-year survival of patients, analysis the relevance of 5-year survival rate with CD55 high expression, ALDH1 expression and clinical disease characteristics, then use Cox proportional analysis model for single-factor analysis of breast cancer patients prognosis, on the basis of single-factor analysis use the multi-factor non-conditional Logistic regression analysis make further filter for the independent risk factors of breast cancer prognosis.
Results:
1 In normal breast tissue, expression of ALDH1 was negative or very weak positive, the expression rate of ALDH1 in breast cancer tissue specimens was 24.6% (16/65); expression of CD55 is low in normal breast tissue, in breast cancer tissue, high expression of CD55 specimens accounted for 33.8% (22/65).
2 Expression of ALDH1 and high expression of CD55 are closely related to recurrence of the tumor(P<0.01), but they are not related with other clinical disease characteristics, such as postmenopausal status, tumor histological type, histological grade, tumor size, lymph node metastasis status, estrogen receptor and progesterone receptor expression, her2 expression, accept other forms of treatment (P>0.05).
3 5-year survival rate of patients which expression of ALDH1, high expression of CD55 was significantly decreased when compared with patients which ALDH1 negative, CD55 low expression (P<0.001).
4 Expression of ALDH1, high expression of CD55, tumor diameter, histological grade, her2 negative expression, patients who did not receive treatment, they are related with 5-year survival rate of breast cancer patients, they are independent factors of breast cancer prognosis.
Conclusion: Expression of ALDH1 and high expression of CD55 are both closely related to recurrence of the tumor and are not related with other clinical disease characteristics of breast cancer patients. They are independent prognostic factors of breast cancer patients.
Part 2 CSC enrich of mouse breast cancer cell line 4T1 and the animal model which enriched with CSC was established
Objective: Use serum-free suspension mammosphere culture and chemotherapy way to enrich breast cancer CSC from 4T1 cell lines in vitro and in vivo respectively, from that cell populations and mouse model which enriched with breast cancer CSC were getted. This foundation can use for anti-breast cancer CSC therapy.
Methods:
1 Mammosphere culture: 4T1 cells were inoculated in serum-free medium(SFM) with cell density of 2×104/ml, change medium with centrifuge after 3 days, when the cell cultured to mammosphere with 100 to 200 cells, digestion with diluted trypsin-EDTA and passage, about 6 to 7 days passage one generation.
2 The establish of mouse model which enriched with breast cancer CSC: 0.2ml 4T1 cell suspension (1.5×106 cells) inoculated in the right shoulder of mouse subcutaneously. Observe the formation of tumor , when the tumor grows about 0.1cm, give the mouse 0.2mg/ml 5-FU 0.2ml intraperitoneally once a day. Seven days later, the administration way was changed to once a week. Four weeks later the mouse were sacrificed. Sterile removal mouse tumor, the tumor was grinded with organize grinders and make cell suspension, adjust the living cell concentration to 7.5×106/ml with PBS. Repeat the above procedure make the second generation mouse model, four generations of mouse model were making at last.
3 CSC detection: CD44+CD24-/low cell content was detected with flow cytometry, non-fluorescent dye staining side population(SP) cell ratio were counted after cells stained with HOECHST33342. MDR1, BCRP, ALDH1 mRNA expression in enriched CSC cells were detected with fluorescence quantitative PCR. CD55 and ALDH1 protein expression in tumor tissue were detected with immunohistochemistry. The tumorigenicity of the enriched CSC was detected with tumorigenicity test in mouse.
Results:
1 4T1 cells in SFM can form continuous passage mammosphere, In 10th generation mammosphere, the content of CD44+CD24-/low cell was 68.9±3.78%, SP cell ratio was 56.1±5.18%, MDR1, BCRP, and ALDH1 mRNA expression was compared with the control cells 4T1 cultured in SSM raised for 1.63±0.21 fold, 2.14±0.21 fold, 3.41±0.53 fold respectively. Tumorigenic test showed that 2×103 mammosphere cells can form tumor in mice, whereas the control 4T1 cells cultured in SSM requires at least 2×105 cells to form tumor.
2 Mouse model enriched with breast cancer CSC has established with 5-Fu. In the third generation of mouse tumor, CD44+CD24-/low cell ratio was 69.0±1.6%, SP cell ratio was 61.3±2.6%, MDR1, BCRP, and ALDH1 mRNA expression was raised by 4.35±0.21 fold 6.14±0.47 fold, 3.78±0.32 fold, compared with the control of the tumor tissue(P <0.01). ALDH1 expression was positive, CD55 strong positive cell ratio was 17.3±1.9%, they express much stronger than the control tumor tissue. Tumorigenicity test showed that 2×103 mouse model cells can form tumor, whereas the control tumor requires at least 2×105 cells to form tumor.
Conclusion: The CSC induced for 4T1 cell line with serum-free suspension mammosphere culture in vitro, the mouse model which enriched CSC with chemotherapy drugs was established. Through a number of target detection and tumorigenicity test we confirmed that the way we used in this study was successful for the enrich of breast cancer CSC. This study provides basis for further research target killing CSC.
Part 3 Research for Parthenolide activity of mouse breast cancer CSC
Objective: To investigate the effects of Parthenolide(PTL) to breast cancer CSC in vivo and in vitro, provide basis for the treatment of breast cancer with PTL on clinical.
Method:
1 Effect of PTL to kill CSC from 4T1 cell lines: the 10th generation of mammosphere cells inoculated in 25ml flasks with the cell concentration 5×104/mL in SFM culture medium, 25 bottles cells were randomly divided into control group, 5-FU group, PTL group, 5-FU+PTL group, PTL+NAC(N-acetyl cysteine)Group,altogether 5 groups. Control group adds normal saline 20μl, 5-FU group adds 0.1mg/ml 5-FU20μl, PTL group adds 1mg/ml PTL 20μl, 5-FU+PTL group adds 0.2mg/ml 5-FU 10μl and 2mg/ml PTL 10μl, PTL+NAC group adds 2mg/ml PTL 10μl and 20mg/ml NAC 10μl. After culture 48 hours, centrifuged and changed to SFM for continued culture. After 7 days, the cells were observed for the mammosphere forming and then cells were collected for further testing.
2 Experiment of PTL kill CSC in mouse model: 25 female Balb/c mice were randomly divided into control group, 5-FU group, PTL group, 5-FU+PTL group, PTL+NAC group. Sterile removal mouse tumor of the third generation mouse model(Part 2, 2.1), the tumor was grinded with organize grinders and make cell suspension. Absorb 0.2ml cell suspension (containing 1.5×106 cells) were inoculated subcutaneously. 24 hours after inoculation, control group mice were given normal saline 0.4ml, intra-abdominal 0.2ml and the tumor cell inoculation site 0.2ml respectively. 5-FU group mice were given 0.2mg/ml of 5-FU 0.4ml, PTL group were given 1mg/ml of PTL 0.4ml, 5-FU+PTL group were given 0.2mg/ml 5-FU and 1mg/ml PTL mixture 0.4ml, PTL+NAC group were treated with 1mg/ml PTL and 10mg/ml NAC mixture 0.4ml, the drugs given site and dose were identified with control group. Time for given drugs were once a day, sharing seven days, seven days later changed to once a week. Four weeks later, the mice were sacrificed, tumors were taken for testing.
3 The residual CSC detection after PTL killed: CD44+CD24-/low cell content was detected with flow cytometry, non-fluorescent dye staining side population(SP) cells ratio were counted after cells stained with HOECHST33342, MDR1, BCRP, ALDH1 mRNA expression were detected with fluorescence quantitative PCR. CD55 and ALDH1 protein expression in tumor tissue were detected with immunohistochemistry.
Results:
1 PTL killed CSC from 4T1 cell lines in vitro: After 5-FU, PTL, 5-FU+PTL or PTL+NAC treatment and cultured in SFM, control group, 5-FU group, PTL+NAC group can form mammospheres clearly, PTL group formed only fewer mammospheres in SFM, there was only scattered mammospheres existence, 5-FU+PTL group live cells were too little, the cells were lysised due to apoptosis. The CD44+CD24-/low cell contents of control group, 5-FU group, PTL group, PTL+NAC group were 71.2±2.3%, 75.6±3.1%, 12.3±1.9%, 58.1±2.6%, respectively; non-fluorescent staining(SP) cells percentages were 56.7±3.4%, 62.0±2.7%, 8.1±1.1%, 51.5±2.3%, respectively, PTL group was significantly lower than the control group, 5-FU group and PTL+NAC group(P<0.01).
2 Animal experiment for PTL killed CSC in mouse model: After 5-FU, PTL, 5-FU+PTL or PTL+NAC treatment, the velocity and diameter of the tumor forming in control group and 5-FU group are much larger than PTL group, 5-FU+PTL group, PTL+NAC group(P<0.01). The CD44+CD24-/low cell contents in tumor of control group, 5-FU group, PTL group, 5-FU+PTL group, PTL+NAC group were 57.3±4.1%, 68.7±3.2%, 42.5±3.7%, 39.1±2.9%, 50.1±3.1%, respectively; non-staining(SP) cell percentages were 58.1±4.1%, 61.3±2.6%, 36.9±3.5%, 39.2±1.8%, 42.7±2.5%, respectively. Both CD44+CD24-/low cell contents and SP cell percentages, PTL group, 5-FU+PTL group, PTL+NAC group were significantly lower than control group, 5-FU group (P<0.05). MDR1 gene expression in tumor tissue for 5-FU group, PTL group, 5-FU+PTL group, PTL + NAC group compared with control group was 1.11±0.11 fold, 0.92±0.12 fold, 1.03±0.15 fold, 0.94±0.08 fold, all the groups have no significant difference(P>0.05); BCRP gene expression in tumor tissue for 5-FU group, PTL group, 5-FU+PTL group, PTL + NAC group compared with control group was 1.09±0.16 fold, 0.97±0.10 fold, 1.13±0.11 fold, 0.95±0.13 fold, all the groups have no significant difference (P>0.05). ALDH1 gene expression in tumor tissue for 5-FU group, PTL group, 5-FU+PTL group, PTL + NAC group compared with control group was 1.16±0.12 fold, 0.38±0.07 fold, 0.42±0.09 fold, 0.57±0.04 fold. PTL group, 5-FU+PTL group, PTL+NAC group were significantly lower than control group and 5-FU group (P<0.05).
Conclusions: PTL could target to breast cancer CSC, change the state of CSC, so CSC contents in the cultured mamosphere cells or tumor tissue significantly reduced. The roles of PTL targeting CSC is mainly due to oxidative stress. Anti-oxidant drug NAC can clearly antagonistic the effect in vitro, but in mice it is not obvious. Combination with other chemotherapy drugs PTL may enhance the effects of other chemotherapy drugs; PTL could be used as a chemotherapy sensitizer.
引文
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