【作者】 张书锋；

【导师】 张汉明； 陈万生； 李润平；

【作者基本信息】 第二军医大学 ， 生药学， 2008， 博士

【摘要】 化疗具有许多副作用,如脱发、恶心、呕吐和腹泻。大多数传统的细胞毒抗肿瘤药物伤害迅速分裂的毛囊细胞而导致脱发。化疗脱发（Chemotherapy-induced alopecia,CIA）是身体上和心理上最令人痛苦的肿瘤化疗药物副作用之一,也是临床肿瘤学主要的未解决难题之一。绿茶多酚（GTP）主要由表没食子儿茶素没食子酸酯[（-）-epigallocatechin-3-gallate（EGCG）]、表没食子儿茶素[（-）-epigallocatechin（EGC）]、表儿茶素没食子酸酯[（-）-epicatechin-3-gallate（ECG）]、表儿茶素[（-）-epicatechin（EC）]、没食子儿茶素[（+）-gallocatechin（GC）]和儿茶素（C）组成。以前研究证明,茶多酚能促进小鼠正常毛发的再生,EGCG在体外可通过对真皮乳头细胞的增殖和抗凋亡作用而促进人毛发的生长,并且可能通过选择性地抑制5α-还原酶的活性而有助于预防或治疗雄激素性秃发。在我们的前期工作中,发现茶多酚可能对临床化疗脱发有效,并在小鼠化疗脱发模型中证明口服茶多酚能显著减轻环磷酰胺引起的脱发并促进毛发再生。但至今未见其它有关茶多酚及其成分对化疗脱发作用的报道。本课题进一步研究茶多酚在小鼠和大鼠化疗脱发模型中对环磷酰胺引起脱发的影响及其作用机理;研究茶多酚在体外对毛囊生长和阿霉素诱导的毛囊及毛囊细胞毒性的影响;研究茶多酚对化疗药物抗肿瘤疗效及毒性的影响。用C57BL/6小鼠化疗脱发模型研究茶多酚对环磷酰胺引起脱发的影响。脱毛后第9天,小鼠一次性腹腔注射环磷酰胺100～120 mg/kg诱导脱发。脱毛后第13和第20天,与环磷酰胺组相比,环磷酰胺+茶多酚组小鼠背部皮肤脱发百分比明显减小。结果表明,茶多酚能显著减少环磷酰胺诱导的脱发,并能促进毛发的再生。用新生大鼠化疗脱发模型研究茶多酚对环磷酰胺诱导脱发的影响。出生后第13天大鼠一次性腹腔注射环磷酰胺35 mg/kg诱导脱发。注射环磷酰胺后第5天,环磷酰胺组大鼠出现轻微或中度严重脱发,第8天大鼠全身脱发。注射环磷酰胺后第5天,茶多酚（100 mg/kg,i.p.）+环磷酰胺组大鼠出现可察觉的脱发,第8天大多数出现轻微脱发。可见茶多酚（100 mg/kg,i.p.）对环磷酰胺引起的新生大鼠脱发具有明显的预防作用。注射环磷酰胺后第8天,与环磷酰胺组相比,茶多酚+环磷酰胺组大鼠背部毛囊的凋亡细胞明显减少,并且多数毛囊仍处于生长期。这表明茶多酚可明显减轻环磷酰胺诱导的毛囊细胞凋亡,阻止毛囊退行并延长毛发生长期持续时间。以添加10μg/ml胰岛素、100 U/ml青霉素、100μg/ml链霉素的William’s E培养基培养小鼠和新生大鼠触须毛囊。将小鼠触须毛囊培养5天,0.1～1000μg/ml茶多酚各组毛囊毛发的平均生长长度呈剂量依赖性减少,其中10～1000μg/ml茶多酚处理的毛囊,其平均毛发生长长度明显小于对照组。将大鼠触须毛囊培养5天,0.1～1000μg/ml茶多酚各组毛囊毛发的平均生长长度呈剂量依赖性减小,其中10～1000μg/ml茶多酚处理的毛囊,其平均毛发生长长度明显小于对照组;10^-9～10^-3μg/ml茶多酚处理的毛囊,其平均毛发生长长度大于对照组,其中10^-510^-3μg/ml茶多酚处理的毛囊,其平均毛发生长长度明显大于对照组。结果表明茶多酚对毛囊毛发的生长具有促进或抑制作用,即较低浓度的茶多酚能促进毛囊的生长,较高浓度茶多酚抑制毛囊的生长。用毛囊和毛囊细胞培养研究茶多酚在体外对阿霉素诱导的毛囊及毛囊细胞毒性的影响。0.1、1和10μg/ml茶多酚处理的毛囊,其平均毛发生长长度明显大于阿霉素组,而高浓度（≥100μg/ml）茶多酚处理的毛囊,其平均毛发生长长度与阿霉素组相比差异不明显。可见,在体外0.1和10μg/ml茶多酚显著抑制阿霉素对毛囊的毒性,而高浓度茶多酚（≥100μg/ml）对毛囊不具有保护作用。与阿霉素处理的毛囊真皮鞘细胞相比,1和10μg/ml茶多酚处理的真皮鞘细胞的细胞活力显著增加。这表明1和10μg/ml的茶多酚显著抑制阿霉素对毛囊细胞的毒性。用阿霉素或阿霉素+茶多酚培养的大鼠触须皮肤的切片进行H&E和TUNEL染色检测,与阿霉素组毛囊相比,1μg/mi茶多酚+10μg/ml阿霉素组毛囊球部凋亡细胞明显减少。这表明茶多酚可明显减轻阿霉素诱导的毛囊细胞凋亡。在毛囊细胞集落形成检测中,29.30%ADM+TP处理的毛囊及9.30%ADM处理的毛囊形成毛囊细胞集落,并且ADM+TP处理毛囊形成的毛囊细胞集落的细胞数明显多于ADM处理的毛囊。这表明茶多酚可明显抑制阿霉素对毛囊的毒性。用小鼠S₁₈₀模型研究茶多酚对环磷酰胺疗效及荷瘤小鼠外周血象的影响。茶多酚150、300、600 mg/kg单用,3个剂量组的平均瘤重均低于对照组,其中低剂量组与对照组相比有显著性差异。环磷酰胺与茶多酚各剂量组合用与环磷酰胺单用相比,瘤重虽有所降低,但差异不明显。结果表明较低浓度的茶多酚具有显著的抗肿瘤作用,并且茶多酚不影响环磷酰胺的抗肿瘤疗效。150、300、600 mg/kg茶多酚组的白细胞数均明显高于对照组。环磷酰胺+茶多酚各剂量组的白细胞、红细胞、血小板数及血红蛋白含量与环磷酰胺组相比,差异均不明显。结果表明各剂量茶多酚单用对荷瘤小鼠具有明显的升高白细胞作用,但与环磷酰胺合用后对环磷酰胺引起的荷瘤小鼠外周血象降低没有明显的改善作用,这表明150～600 mg/kg茶多酚对荷瘤小鼠应用环磷酰胺后的骨髓抑制毒性没有明显的改善作用。用人HepG2肿瘤细胞培养研究茶多酚在体外对阿霉素抗肿瘤作用的影响。在体外,阿霉素和茶多酚对HepG2细胞均具有细胞毒作用,而且茶多酚与阿霉素合用不影响阿霉素的细胞毒性。用白细胞减少症模型研究茶多酚在小鼠体内对环磷酰胺导致毒性的影响。与给药前相比,第7天环磷酰胺组和环磷酰胺+茶多酚组的白细胞总数均显著下降,但后来白细胞总数逐渐回升,第15天环磷酰胺+茶多酚组的白细胞总数明显高于环磷酰胺组,表明茶多酚具有明显的升白作用。综上所述,茶多酚在小鼠和大鼠化疗脱发模型中对环磷酰胺引起的脱发具有显著的预防作用;在大鼠化疗脱发模型中,茶多酚可明显减轻环磷酰胺诱导的毛囊细胞凋亡,阻止毛囊退行并延长毛发生长期持续时间:茶多酚在体外对毛囊毛发的生长具有促进或抑制作用:茶多酚在体外能显著减轻阿霉素诱导的毛囊细胞凋亡,并显著减轻阿霉素对毛囊和毛囊细胞的毒性;茶多酚在小鼠体内具有显著的抗肿瘤作用,且与环磷酰胺合用不影响环磷酰胺的抗肿瘤疗效:茶多酚对环磷酰胺引起的正常小鼠的白细胞降低具有显著的抑制作用,且对S₁₃₀荷瘤小鼠的白细胞有显著的提升作用,但对环磷酰胺引起的S₁₈₀荷瘤小鼠外周白细胞降低没有明显的抑制作用;此外,茶多酚在体外与阿霉素合用不影响阿霉素的细胞毒性。因此,茶多酚可能有望开发成为一种化疗脱发预防药物,预防机理可能与其对毛囊细胞增殖的抑制作用和减轻毛囊细胞凋亡有关,需从细胞和分子水平进一步进行研究。更多还原

【Abstract】 Chemotherapy has many side-effects like hair loss,nausea,vomiting,and diarrhea.Most traditional cytotoxic anticancer drugs attack rapidly dividing hair follicle cells and induce alopecia（hair loss）.Chemotherapy-induced alopecia（CIA） is one of the most physically and psychologically distressing side effects of cancer chemotherapeutic drugs and represents one of the major unresolved problems of clinical oncology.Green tea polyphenols（GTP） consist mainly of（-）-epigallocatechin-3-gallate（EGCG）, （-）-epigallocatechin（EGC）,（-）-epicatechin-3-gallate（ECG）,（-）-epicatechin（EC）, （+）-gallocatechin（GC） and catechin（C）.Previous investigations demonstrated that oral feeding of GTP significantly increased normal hair re-growth in mice,and EGCG promoted hair growth and the proliferation of cultured dermal papilla cells in vitro,and might be useful in the prevention or treatment of androgenetic alopecia by selectively inhibiting 5α-reductase activity.In our previous work,it was found that tea polyphenols（TP） might be effective to human CIA,and oral application of TP was found to significantly decrease cyclophosphamide（CYP）-induced alopecia and increase hair re-growth in a mouse CIA model.However,no other report has been issued to date on the effect of tea polyphenos on CIA.This study was undertaken to further evaluate the effect of TP on CYP-induced alopecia in the mouse and rat CIA models and its mechanism of action;to investigate its effect on hair follicle growth,ADM-induced toxicity to hair follicles and dermal sheath cells（DSCs） in vitro,and to study its effect on the anticancer activity and toxicity of chemotherapeutic drugs.C57BL/6 mouse CIA model was used to investigate the effect of TP on CYP-induced alopecia.Alopecia was induced by a single intraperitoneal（i.p.） injection of CYP （100～120 mg/kg） on mice on day 9 after depilation.Fourteen or 20 days after depilation, the percentage of alopecic back skin in TP treated mice was significantly decreased compared with CYP treated alone,indicating that oral application of TP significantly reduced CYP-induced alopecia and increased hair regrowth in the mouse CIA model.A neonatal rat CIA model was used to investigate the effect of TP on CYP-induced alopecia.Alopecia was induced by a single i.p.injection of CYP（35 mg/kg） on 13 day-old rats.Eight days after CYP injection,most of the TP（100 mg/kg,i.p.） treated rats had mild alopecia with less than 50%hair loss,while CYP treated alone rats had total alopecia, indicating that i.p.injection of 100 mg/kg TP significantly prevented CYP-induced alopecia in the newborn rat CIA model.On day 8 after CYP injection,the apoptotic cells in the hair follicles of back skin of TP+CYP treated rats were obviously reduced compared with that of CYP treated ones,and most hair follicles were in anagen stage of hair cycle. This indicated that TP markedly alleviated CYP-induced apoptosis in hair follicle cells, prevented hair follicle regression and increased anagen duration.Mouse and neonatal rat vibrissa follicles were cultured in William’ medium E supplemented with 10 mg/l insulin,50 U/ml of penicillin and 50μg/ml of streptomycin. The increase in hair fiber length in 0.1～1000μg/ml TP-treated groups decreased in a concentration-dependent manner,and that in the 10～1000μg/ml TP-treated ones significantly increased compared with the vehicle-treated control.On the other hand,the increase in hair fiber length in 10^-9～10^-3μg/ml TP-treated groups increased,and 10^-5～10^-3μg/ml TP-treated groups significantly increased compared with the vehicle-treated control. The results indicated that higher concentrations of TP inhibited the growth of hair follicles and lower concentrations of TP increased the growth of hair follicles in vitro.Hair follicle and hair follicle cell culture models were used to investigate the in vitro effect of TP on ADM-induced toxicity to hair follicles and hair follicle cells.The hair fiber length of 0.1,1 and 10μg/ml TP-treated hair follicles increased significantly compared with ADM-treated ones,indicating that 0.1,1 and 10μg/ml TP significantly alleviated ADM-induced toxicity to hair follicles in vitro.1 and 10μg/ml TP significantly increased the cell viability of DSCs compared with the ADM-treated control,indicating that 1 and 10μg/ml TP significantly alleviated ADM-induced toxicity to DSCs.Tissue sections from vibrissa skin of rats after incubation with ADM or ADM plus TP were examined with hematoxylin and eosin（H&E） staining and Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling（TUNEL） staining.The apoptotic cells in the hair bulb of TP+AMD treated hair follicles were obviously reduced compared to that of AMD treated ones.This indicated that TP markedly alleviated AMD-induced apoptosis in hair follicle cells.In a cell colony-forming assay,hair follicle cell colonies were formed from 29.30%ADM+TP-treated hair follicles and 9.30%ADM-treated ones,and the cell number of colonies formed from ADM+TP-treated hair follicles was greater than that from ADM-treated ones.This indicated that TP marked inhibited ADM-induced toxicity to hair follicles.In a mouse model of S₁₈₀ tumor,TP（150 mg/kg）,or CYP（30 mg/kg） combined with TP （150,300 or 600 mg/kg） inhibited the growth of S₁₈₀ significantly compared with untreated control,and CYP combined with TP（150,300 or 600 mg/kg） also enhanced the anti-tumor activity compared with CYP alone;however the differences were not statistically significant.Meanwhile,TP（150,300 or 600 mg/kg） co-administrated with CYP did not increase total white blood cell（WBC） count significantly compared with CYP alone, although TP（150,300 or 600 mg/kg） increased total WBC count,and TP（150 mg/kg） increased total WBC count significantly compared with untreated control.The results indicated that TP inhibited the growth of S₁₈₀ and did not affect the anti-tumor activity of CYP,but did not reduce CYP-induced leucopenia in S₁₈₀-bearing mice.Human HepG2 cell culture model was used to investigate the effect of TP on the antitumor activity of ADM.In vitro,ADM and TP had cytotoxic effect on HepG2 cells, and combination of TP and ADM did not affect the cytotoxicity of ADM.In a mouse model of CYP-induced leucopenia,there was a significant decrease in the total white blood cell（WBC） count of both CYP alone and CYP along with the TP treated animals on 7th day,but later total WBC count was found to be significantly higher in TP treated group.The results indicated that oral administration of TP significantly reduced CYP-induced leucopenia in normal mice.In summary,this study demonstrated that TP significantly prevented CYP-induced alopecia in the mouse and rat CIA models.In the rat CIA model,TP markedly alleviated CYP-induced apoptosis in hair follicle cells,prevented hair follicle regression and increased anagen duration.TP inhibited or increased the growth of hair follicles in vitro; TP significantly alleviated ADM-induced apoptosis in DSCs and ADM-induced toxicity to hair follicles and DSCs in vitro.TP did not affect the anticancer activity of CYP in mice, but did not reduce CYP-induced leucopenia in S₁₈₀-bearing mice although it significantly reduced CYP-induced leucopenia in normal mice and increased total WBC count in S₁₈₀-bearing mice;Furthermore,combination of TP and ADM did not affect the cytotoxicity of ADM in vitro.Thus,TP may be a promising drug for the prevention of CIA, and the mechanism of prevention may be related to its inhibition of the proliferation of keratinocytes of hair follicles and alleviation of apoptosis,the cellular and molecular mechanism remains to be further investigated.更多还原