摘要
目的
骨肉瘤是骨的原发恶性肿瘤第一位恶性肿瘤,约占骨的原发恶性肿瘤的20%。其具有恶性程度高,好发于青少年、易复发和转移、死亡的特点。随着新辅助化疗的出现,手术技术的改进,大部分骨肉瘤病人可以保肢治疗,5年生存率可达到70%。以甲氨蝶呤、阿霉素、异环磷酰胺及顺铂为主要化疗药物对骨肉瘤细胞杀伤抑制作用已经得到认可并且广泛使用。但是,化疗也随之带来患者免疫力下降、抑制造血系统、恶心呕吐以及肝肾心脏功能损伤等不良反应,同时也出现化疗耐药等不良后果,导致部分病人无法完成化疗,影响患者生存质量及寿命。
因此,许多其它治疗骨肉瘤的手段出现,包括放疗、基因治疗、免疫治疗以及分子靶向治疗等多种新形的治疗方式。其中生物免疫治疗肿瘤已成为治疗肿瘤的重要措施,它通过激发和利用机体免疫反应来拮抗肿瘤细胞,调节与平衡机体免疫功能,分化和抑制肿瘤细胞生长,并有选择性杀伤肿瘤细胞的作用。目前以免疫活性细胞的过继免疫治疗是最引人注目的手段之一。在过继免疫治疗的免疫活性细胞中CIK (eytokine-indueed killer)细胞被认为是最有希望的细胞因子诱导杀伤细胞。它具有增殖能力强、杀瘤活性高、杀瘤谱广、并用对正常骨髓细胞毒性作用小等特点,它对于促进病人免疫系统的重建、骨髓净化以及微小残留病灶的清除等均具有重要的意义。已在临床上应用于肺癌、肝癌、肾癌的治疗,但其与骨肉瘤之间的作用关系目前尚不明确。我们希望通过研究CIK细胞作用骨肉瘤细胞以及作用于骨肉瘤病人,了解其是否具有杀伤骨肉瘤细胞作用,为CIK细胞在骨肉瘤治疗方面提供依据。
中医认为骨肉瘤病机属于本虚标实,由于肝肾亏虚,以肾虚为基本,邪客留滞,气滞血瘀,邪毒积聚成块所形成。因此中医治法以补益肝肾为基本,而六味地黄汤补益肝肾代表方。现代药理研究证实,六味地黄汤能增强机体的细胞免疫和体液免疫。我们研究六味地黄丸中药血清作用耐阿霉素骨肉瘤细胞,了解其是否具有杀伤抑制耐阿霉素骨肉瘤细胞作用,为六味地黄丸的临床应用提供实验基础。
临床上我们用鹿角胶、当归、附子及黄芪组方。具有补益肝肾,益气补血功效,其中组成的单药已有大量报道具有减轻化疗药物对造血系统抑制的作用。但是没有相关报道鹿角胶、当归、附子及黄芪组方减轻化疗不良反应,我们希望通过临床观察检测血液分析,了解我们补肝肾方药是否能够减轻化疗导致的造血系统抑制不良反应。
实验方面：①通过将耐阿霉素骨肉瘤细胞进行培养、传代,了解骨肉瘤细胞生长规律；将第三代骨肉瘤细胞以1×10^7/mL浓度注入裸鼠颈背部皮肤下lml进行骨肉瘤造模,观察造模成功率,在此基础上观察裸鼠成瘤情况,提供一种简单易行的造模方式,为骨肉瘤的研究提供基础。②用六味地黄丸给SD大鼠灌胃,通过腹腔静脉采血,制备含六味地黄丸中药血清,用于作用骨肉瘤细胞。③分离人体外周血单核细胞,采取作者静脉血50ml,经淋巴分离液分离,经离心机离心后,抽取中间单核细胞层,通过第0天加入浓度为100U/mL的IFN-γ。放入37℃,5%C02培养箱中培养24小时后,加入浓度为50 ng/mL CD3、100U/mL的IL1-a和300U/mL IL-2,诱导CIK细胞形成,通过流式细胞仪检测其表型。④分别研究PBS液,含10%六味地黄丸中药血清培养液及1640培养液三者作用耐阿霉素骨肉瘤细胞；研究PBS液、含10%胎牛血清高糖DMEM培养液、含400ug/ml阿霉素0.9%氯化钠溶液、含200ug/ml阿霉素0.9%氯化钠溶液、含100ug/ml阿霉素0.9%氯化钠溶液、含50ug/ml阿霉素0.9%氯化钠溶液及含25ug/ml阿霉素0.9%氯化钠溶液作用耐阿霉素骨肉瘤细胞；研究六味地黄丸中药血清联合不同浓度阿霉素作用骨肉瘤细胞；研究CIK细胞溶液(与骨肉瘤细胞为1：1)、含10%六味地黄丸中药血清培养液、含400ug/ml阿霉素0.9%氯化钠溶液,CIK细胞溶液十含400ug/ml阿霉素0.9%氯化钠溶液、CIK细胞溶液(与骨肉瘤细胞为1：1)十含10%六味地黄丸中药血清培养液及PBS溶液作用体外培养的耐阿霉素骨肉瘤细胞；研究六味地黄丸中药血清联合不同比例CIK细胞作用骨肉瘤细胞。将以上所得的结果进行方差分析。
临床方面：①我们选取10名健康成年人及10位成年骨肉瘤患者外周血各50ml,体外扩增出来骨肉瘤患者和正常献血者的CIK细胞。通过流式细胞仪检测对比正常人与骨肉瘤患者自身诱导出的CIK细胞增殖同FACS表型有何差异,同时比较两组CIK细胞抗骨肉瘤细胞株KH-OS和骨肉瘤耐药性细胞株MG63/ADM的细胞毒性活性。②选取广州中医药大学第一附属医院2009年1月至2011年3月三骨科住院病人确诊为骨肉瘤患者。共有10人纳入,7男3女,年龄12-21岁。实验组6人,对照组4人。观察服用以鹿角胶、附子、当归及黄芪组成的温阳补肾补血方降低化疗药物对骨髓抑制作用。比较两组的WBC、HGB、PLT及NEU变化,将其结果进行t检验。
结果
骨肉瘤细胞生长代谢较普通细胞快,细胞呈梭形不规则,细胞核大,一般3天就增殖7倍。以浓度为1×10^7/ml的骨肉瘤悬液lml注入裸鼠颈背部皮下造模,所有造模裸鼠成瘤,成瘤率100%,经病理切片验证所形成的肿物为接种的骨肉瘤细胞。外周静脉血经淋巴分离液以及离心分层后,分成3层,中间层为白色混浊单核细胞层,通过第0天加入浓度为100U/mL的IFN-γ,第一天加入浓度为50ng/mL CD3、100U/mL的ILI-α和300U/mL的IL-2诱导后,成功诱导出CIK细胞,CIK细胞为圆形透亮的细胞,经流式细胞仪检测,较高表达CD3+CD56+。通过统计学分析,含10%六味地黄丸中药血清培养液无明显抑制骨肉瘤细胞生长增殖作用,与其余两组相比较p均小于0.05。含400ug/ml、200ug/ml及100ug/ml阿霉素0.9%氯化钠溶液对骨肉瘤细胞抑制作用比其余两组阿霉素要强(p<0.05),表现出随着浓度增加而表现增强的杀伤作用。六味地黄丸中药血清联合不同浓度阿霉素作用骨肉瘤细胞,各组之间无明显统计学差异(p=0.618)。六味地黄丸中药血清联合不同比例CIK细胞作用骨肉瘤细胞,各组之间有统计学意义(p=0.001),对骨肉瘤的抑制杀伤作用随着CIK细胞与骨肉瘤细胞比例增加而增强,但是我们实验得到当比例是50：1时反而出现抑制减弱。
我们收集10位健康患者及10位骨肉瘤患者外周静脉血,以同样的方法诱导CIK细胞,经流式细胞仪检测。培养到21天时OS-CIK的平均数量加了35.8倍。而ND-CIK的平均数量增加了81.6倍。CD3+CD56+分别从在第七天的6.6%,10.3%到第21天增加到15.9%(P<0.05),24.8%(P<0.05)。而且,CD3+CD56‘在第17天达到最高,而此时CD4+CD25+阳性率也仅为1.31%。OS-CIK、ND-CIK对KH-OS在效靶比为40：1时达到的抑瘤率分别为53.67%、81.23%,而对MG63/ADM在效靶比为40：1时达到的抑瘤率分别为50.74%、65.70%。
10位骨肉瘤患者化疗期间服用补肝肾中药后血液分析结果,化疗前两组的WBC、HGB、PLT、NEU没有明显差别。说明两组在化疗前各项指标无明显统计学差异。化疗结束后1周,两组WBC明显下降,实验组降至2.73×10^9/L,对照组降至2.048×10^9/L,两者经统计学检验后p=0.081。两组HGB均下降,实验组下降幅度稍高于对照组,实验组下降15g/L,对照组下降19g/L。两组NEU均下降。化疗后一个月,两组HGB继续下降,其余指标基本恢复正常。在整个过程中,PLT变化都在正常范围内。
结论
骨肉瘤细胞体外培养具有增殖快的特点,符合临床上骨肉瘤生长速度快特征。以浓度为1×10^7/ml的骨肉瘤细胞在裸鼠颈背部松弛皮下造模,成瘤率100%,此方法简单易行,成瘤率高。阿霉素对耐阿霉素骨肉瘤细胞的杀伤作用随着浓度升高而增强,呈现浓度依赖关系。10%六味地黄丸中药血清对体外培养的耐阿霉素骨肉瘤细胞无名显杀伤抑制作用,反而有利于骨肉瘤细胞增殖,具体作用机制还需要进一步研究,同时也反应出体外实验的有限性和不足,说明我们中药方药的作用机制复杂,可能是通过调节全身机体多脏器来起到治疗作用,体现出中医的整体观念。也为如何更好的研究药方药提供新的思路和方向。CIK细胞通过诱导可以较容易的获得,生长高峰在14-21天,OS-CIK和ND-CIK均有明显的增殖,但是ND-CIK细胞增殖速度远远高于OS-CIK,均高度表达CD3+CD56+。两者其对骨肉瘤细胞的杀伤作用随着浓度的升高而增强。温阳补肾补血方药可以对化疗导致的骨髓抑制有一定的缓解作用,统计学上两组无明显差异,但是从平均数值上,服用温阳补肾补血中药的病人在WBC、HGB下降幅度小于对照组,PLT没有明显下降。可能与我们观察时间有限,病人量少,病人的依从性有关系。此项临床研究为我们研究中医药在减轻化疗毒副作用提供一定的临床依据,我们还需要进一步研究相关中药作用机体的机制。
Objective
Osteosarcoma is a primary malignant bone tumor, accounts for 20% about the primary malignant tumor bone. It has high degree of malignancy, easy to occur in young people, easy to recurrence, metastasis and death. With the emergence of new adjuvant chemotherapy, improved surgical techniques, most osteosarcoma patients can treat with limb salvage, the 5-year survival rate can be as high as seventy percent. The methotrexate, doxorubicin, ifosfamide and cisplatin has been recognized and widely used as the main anti-inhibition of osteosarcoma cells for chemotherapy. However, it can lead to decreased immunity, inhibit the hematopoietic system, nausea, vomiting, and the cardiac, liver and kidney function damage and other adverse reactions, also resistance to chemotherapy occurring. As a result, the patients unable to complete chemotherapy, affecting survival quality and life.
Therefore, many other treatments of osteosarcoma occuring, including radiation therapy, gene therapy, immunotherapy and molecular target therapy and other new forms of treatment. Tumor immune therapy has become an important method for the treatment of cancer, which stimulating the immune response to antagonize the tumor cells, regulate and balance immune function, differentiation, and inhibit tumor cell growth, and selectively kill the tumor cells. Adoptive immunotherapy with Immunocompetent cells is one of the most Striking means. During the adoptive immunotherapy, the CIK (eytokine-indueed killer) cells are considered the most promising cytokine-induced killer cells. It has highly proliferation ability, highly activity to kill tumor, broad spectrum, and little toxicity to normal bone marrow. It can promote the reconstruction of the immune system of patients, bone marrow purification and the removal of minimal residual disease.It has been used in lung Cancer, liver cancer, renal cell carcinoma, but the relationship between osteosarcoma is unclear. We hope that by studying the role of CIK cells and the effect on osteosarcoma patients, see if it has the function anti-osteosarcoma, provide the basis of using the CIK cells to treat with osteosarcoma.
TCM theory holds that the pathogenesis of osteosarcoma is the vir tual standard, due to liver and kidney deficiency, the deficiency of kidney is essential, foreign evils invasion, lead to qi stagnation an d blood stasis. Therefore, Chinese medicine treatment is benefit liv er and kidney and clinical research found that Liu Wei Di Huang Tang is a classic Chinese herbal formula in this righting training. Modern pharmacological studies confirmed that Liu Wei Di Huang Tang can enh ance the body's cellular and humoral immunity. We studied Liuweidihua ngwan serum effect adriamycin-resistant osteosarcoma cells, see if it has the anti-inhibition of adriamycin-resistant osteosarcoma cells.T o provide the experimental basis for clinical application.
Clinically, we use antler glue, angelica, astragalus and rhizoma typhonii, With the effection of tonicing kidney Qi. Each one of thes e has numerous reports can relieve the inhibitory effect on the hemat opoietic system when chemotherapy. However, no relevant reports about this prescription reduce the adverse effects of chemotherapy, we hop e that through clinical observation of blood analysis, whether this p rescription can reduce the inhibition of hematopoietic system caused by chemotherapy side effects.
Methods
Experimental aspects:By culturing, passaging adriamycin-resistan t osteosarcoma cells, understand the growth regulation of the cells. We uesd the third generation of osteosarcoma cells and make the conce ntration was 1×10^7/mL, injected 1ml under the skin at the back of the neck of nude mice for modeling, observed the success rate in nude mice, and to provide a simple way of making model for osteosarco ma research foundation. We Intragastriced SD rats with Liuweidihuangw an and got the abdominal vein blood, prepared Liuweidihuangwan serum, used this serum effecting osteosarcoma cells. We used 50ml blood fro m the author to Separat human peripheral blood mononuclear cells, sep arated by the lymph liquid separation by centrifugation centrifuge to extract the middle layer of mononuclear cells, by adding the concent ration of day 0 100U/mL of IFN-γ. Into 37℃,5% CO2 incubator for 2 4 hours, adding a concentration of 50 ng/mL CD3, 100U/mL of IL1-αand 300U/mL IL-2, induced the formation of CIK cells, by flow cytomet ry To detect the phenotype. To study PBS solution,10% Liuweidihuangwa n serum and 1640 mediums effect the Adriamycin-resistant osteosarcoma cells. To study PBS solution,10% FBS high glucose DMEM medium, cont aining 400ug/ml ADM 0.9% sodium chloride solution,200ug/ml ADM 0.9% sodium, 100ug/ml ADM 0.9% sodium,50ug/ml ADM 0.9% sodium and 25ug/ml ADM 0.9% sodium effect the Adriamycin-resistant osteosarcoma cells. To study Liuweidihuangwan serum combined with different concentration s of doxorubicin effct Adriamycin-resistant osteosarcoma cells. To st udy CIK cells (1:1 with Adriamycin-resistant osteosarcoma cells), con taining 10% Liuweidihuangwan serum medium, containing doxorubicin 400 ug/ml 0.9% sodium, CIK cells combined with containing 400ug/ml doxoru bicin solution, CIK cells in solution containing 10% Liuweidihuangwan serum, PBS solution effect Adriamycin-resistant osteosarcoma cells. To study Liuweidihuangwan serum combined with different proportion CI K cells with Adriamycin-resistant osteosarcoma cells. The results of the above analysis of variance.
Clinical aspects:We selected 10 healthy adults and 10 adult patients with osteosarcoma, got 50ml blood of each one, amplified the CIK cells in vitro.Comparison the FACS phenotype differences by flow cytometry in normal people and osteosarcoma patients with self-induced of CIK cells, to compare the activity of two groups of CIK cells against KH-OS and MG63/ADM. Select the patients (from January 2009 to March 2011) from Joint Orthopaedic in the first affiliated hospital of Guangzhou University of Traditional Chinese Medicine, diagnosed with osteosarcoma. Total of 10 people included,7 men and 3 women, aged from 12 to 21 years.6 people joined the experimental group and 4 patients jinned control group. To observe the effect of reducing the bone marrow suppression of chemotherapy after taking the decoction (antler glue, angelica, astragalus and rhizoma typhonii). Comparing the two groups in WBC, HGB, PLT, LYM%, and NEU%, using t test of its results.
Result
The adriamycin-resistant osteosarcoma cells growth and metabolism faster than normal cells, cell morphology were fusiform, irregular, large nuclei, its proliferated to 7 times only 3 days. The nude modeling which used the concentration of osteosarcoma cells was 1×10-7/mL, injected lml under the skin at the back of the neck of nude mice. The tumor rate was 100%, the pathology report the tumors were the osteosarcoma. Separation of peripheral blood by the lymphatic fluid and centrifuged stratified, divided into 3 layers, the middle layer is white cloudy mononuclear cell layer, by adding the concentration of day 0 100U/mL of IFN-γ. Into 37℃,5% CO2 incubator for 24 hours, adding a concentration of 50 ng/mL CD3, 100U/mL of IL1-αand 300U/mL IL-2, induced the formation of CIK cells, successfully induced CIK cells CIK cells were round translucent cells which high expression of CD3+CD56+by flow cytometry. Through statistical analysis,10% Liuweidihuangwan serum medium without inhibited the growth of osteosarcoma cell proliferation, when compared with the other two groups its p<0.05. The suppression of osteosarcoma cells 400ug/ml,200ug/ml and 100ug/ml 0.9% sodium chloride solution are stronger than the other two groups of doxorubicin (p<0.05), showing enhanced performance with the increase of concentration. When Liu Wei Di Huang Wan serum combined with different concentration of adriamycin effected osteosarcoma cell, there was no significant difference between groups (p=0.618). Liu Wei Di Huang Wan serum associated with different proportions of CIK cells effected the adriamycin-resistant osteosarcoma cells, the result among the groups was statistically significant (p 0.001), the inhibition of the killing effect of osteosarcoma cells showing enhanced performance with the increase of concentration, but When we when the ratio is 50:1(CIK:adriamycin-resistant osteosarcoma cells), it appears less inhibited.
We collected peripheral blood from 10 healthy patients and 10 patients with osteosarcoma, inducing CIK cells by the same way. Culturing to 21 days the average number of OS-CIK 35.8-fold increase and ND-CIK increased 81.6 times. CD3+CD56+, respectively, from 6.6%,10.3% in the seventh day to 15.9% (P<0.05),24.8% in the 21th days (P<0.05). Moreover, CD3+ CD56+in the 17th days reached the highest level at that time when CD4 +CD25+-positive rate only 1.31%. OS-CIK, ND-CIK effected on the KH-OS in the target ratio of 40:1 to achieve the inhibition rates were 53.67%, 81.23%, while the effection on the MG63/ADM achieve the suppression tumors were 50.74%,65.70%.
10 patients with osteosarcoma drinking tonic the kidney yang medicine during chemotherapy and taking blood analysis the results. The WBC, HGB, PLT, NEU among two groups was no significant difference before chemotherapy.1 week after chemotherapy, two groups of WBC decreased, in the experimental group dropped to 2.73×10^9/L, the control group dropped to 2.048×10^9/L(p= 0.081). HGB both groups decreased, the experimental group dropped 15g/L and the control group was decreased by 19g/L. NEU in both groups decreased.1 month after chemotherapy, both groups continued to decline HGB, the other indexes returned to normal. Throughout the process, PLT are within the normal range.
Conclusion
Osteosarcoma cells in vitro with the features of fast proliferati on, in line with the clinical features of osteosarcoma fast growth. T he uesd osteosarcoma cells and make the concentration was 1×10^7/mL, injected lml under the skin at the back of the neck of nude mice for modeling, this is a simple way to modeling and high successfull rate s. The killing consequent of adriamycin effected adriamycin-resistant osteosarcoma cells was enhanced with the concentration, showing a co ncentration-dependent relationship.10% Liuweidihuangwan serum medium without inhibited the growth of osteosarcoma cell proliferation, but is conducive to the proliferation of osteosarcoma cells, the specifi c mechanisms still need further research. This reflect the limitation of nature in vitro and inadequate to illustrate the mechanism of ou r Chinese prescription, the mechanism of our Chinese prescription ma ybe by regulating multiple organs of the body to play a role, reflect ing the 1 concept of holism in TCM. This is supply new ideas and new ways for how to better research for prescription drugs. CIK cells ca n be more easily acquisit through the induction, its growth peak at 1 4 to 21 days, OS-CIK and ND-CIK proliferation significantly, but the ND-CIK cells much faster than OS-CIK, were highly expressed in CD3+ CD56+. Both its killing effect of osteosarcoma cells was enhanced as the concentration increased. Our formula can relief bone marrow sup pression caused by chemotherapy, even though no significant differenc e between the two groups statistically, WBC, HGB small decrease in th e control group and PLT no significant drop. The reasons may be relat ed to our limited observation time, less patients and the patient's c ompliance. This clinical study provide a clinical basis for our Tradi tional Chinese Medicine relieve the side effects of chemotherapy, we also need to further study the mechanisms.
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