益肾骨康方治疗骨转移癌痛临床研究及对骨转移瘤信号转导机制作用
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摘要
骨转移癌是晚期恶性肿瘤常见的并发症。骨转移癌的主要症状是疼痛。其治疗目的是减轻症状,提高生活质量和延长生存期,这是临床治疗的难点和热点。西药治疗均有一定的不良反应及成瘾性,不利于药物的合理使用,因此寻找不良反应小、无成瘾性的药物成为治疗骨转移癌的重要方向。中医中药以其毒副作用小,疗效佳在骨转移癌痛的治疗方面发挥重要的作用。本研究在前期临床辨证治疗的基础上,开展补肾化瘀法治疗骨转移癌疼痛的临床研究及其对溶骨性骨转移癌信号转导机制以及镇痛的分子生物学机制的深入研究。本研究分五部分：(1)补肾化瘀中药治疗骨转移癌疼痛的临床研究；(2)建立溶骨性骨转移裸鼠模型评价益肾骨康方抑制骨转移发生的作用；(3)益肾骨康方对溶骨性骨转移癌FAP信号转导机制的调控作用；(4)益肾骨康方对骨转移癌疼痛大鼠脊髓背角的作用；(5)益肾骨康方对OPG/RANKL/RANK系统的调控作用。
第一部分：补肾化瘀中药治疗骨转移癌疼痛的临床研究
[目的]本研究以给患者造成极大痛苦的骨转移癌疼痛为研究对象,对前期研究有确切疗效的补肾化瘀中药进行疗效评价。
[方法]按照临床试验设计规范,采用平行、随机、对照临床研究原则,根据中医辨证标准将符合诊断标准的骨转移癌疼痛患者采用随机法分为治疗组和对照组,共纳入观察病例51例,中药组26例,对照组25例。中药组患者在口服泰勒宁基础上,同时口服补肾化瘀中药“益肾骨康方”,同单纯口服氨酚羟考酮泰勒宁组进行对照。通过对疼痛影响评分、体力状况、疼痛评分、泰勒宁用量等关键性指标进行组间及治疗前后比较,综合分析、科学评价,观察补肾化瘀法辨证治疗对于骨转移癌疼痛患者疼痛的治疗作用。
[结果]
1、对全部入组病例的年龄、各组男女性别比例情况进行基线分析,中药组男患者与女患者的比例分别为46.15%和53.85%,对照组男患者与女患者的比例分别为48%和52%,χ2检验结果示两组男女比例在统计学上无显著性差异(P>0.05)。中药组和对照组年龄的分别为65.15±11.43和63.28±13.48,t检验结果示两组年
龄在统计学上无显著性差异(P>0.05)。
2、通过对两组病例疼痛影响评分有效率比较(例数／百分比),结果显示中药组与对照组的疼痛影响评分有效率分别为65.38%和28%,治疗后两组病例疼痛影响评分有效率比较有显著性差异(P<0.05)。
3、经过14天的治疗,中药组KPS评分显效9例,有效13例,无效4例,有效率达84.61%,而对照组显效4例,有效9例,无效12例,有效率为52.0%。经秩和检验,在14天的治疗后,治疗组患者的生活质量有效率高于对照组,具有统计学差异(P<0.05)。中药组治疗后KPS评分为66.92±12.89与中药组治疗前52.31±13.66相比较统计学上有极显著性差异(P<0.01)。对照组治疗后KPS评分为59.20±13.52与治疗前56.40±11.50相比统计学上无显著性差异(P>0.05)。
4、治疗前两组NRS评分比较,中药组为5.08±1.62,对照组为5.12±1.59,两组在统计学上无显著性差异(P>0.05),说明两组具有可比性；治疗后两组NRS评分都有所降低,中药组为2.73±1.51与治疗前5.08±1.62比较有显著性差异(P<0.05),对照组治疗后为4.16±2.13与治疗前5.12±1.59比较无显著性差异(P>0.05)；治疗后两组之间比较,有显著性差异(P<0.05)。
5、治疗前两组首日泰勒宁单日用量,中药组与对照组分别为6.04±1.82和6.56±2.31,两者比较无显著性差异(P>0.05),说明两组具有可比性；治疗后两组泰勒宁单日用量都有所降低,治疗组治疗后泰勒宁单日用量为4.35±0.63,与治疗前比较统计学上有显著性差异(P<0.05),对照组治疗后泰勒宁单日用量5.60±2.06与治疗前比较无显著性差异(P>0.05)；治疗后两组之间比较,有显著性差异(P<0.05)。
6、安全性评价：在本试验中全部患者治疗前后均行肝功、肾功、血常规、心电图检查,治疗前后统计结果显示无明显变化,提示补肾化瘀中药使用安全,对人体心、肝、肾、造血系统无明显毒副作用。试验期间,治疗组未发现明显不良反应。提示补肾化瘀中药安全,无明显毒副作用。
[结论]
1、补肾化瘀法治疗骨转移癌疼痛能够提高对疼痛影响评分的有效率；
2、补肾化瘀法治疗骨转移癌疼痛能够提高患者的生存质量；
3、补肾化瘀法治疗骨转移癌疼痛能够降低NRS评分,减轻疼痛水平；
4、补肾化瘀法治疗骨转移癌疼痛能够减少止痛药泰勒宁的单日用量；
5、补肾化瘀法治疗骨转移癌疼痛无明显不良反应,无明显毒副作用,是安全的。
第二部分：建立左心室注射肺腺癌溶骨性骨转移裸鼠模型评价益肾骨康方抑制溶骨性骨转移发生的作用
[目的]本实验以获得专利的具有溶骨性骨转移特性的肺腺癌细胞系通过左心室注射法建立溶骨性骨转移模型,并在此模型基础上观察中药的干预作用。
[方法]本研究采用了获得专利的CPAYANGIBM细胞系,在杨顺芳教授的指导下,采用左心室注射法建立了肺腺癌骨转移裸鼠模型,最终经过Micro SPECT/CT显像和病理切片证实模型组发生了100%的骨转移癌,证实造模成功。将裸鼠随机分为6组,每组6只,并于造模后第3天起给予药物干预。益肾骨康方低剂量组：益肾骨康方浸膏剂混悬液3g/kg,1次/日×5周,灌胃；益肾骨康组中剂量组：益肾骨康方浸膏剂混悬液12g/kg,1次/日×5周,灌胃；益肾骨康组高剂量组：益肾骨康方浸膏剂混悬液24g/kg,1次/日×5周,灌胃；帕米膦酸二钠组：帕米膦酸二钠4.5mg/kg,2次/周×5周皮下注射给药；益肾骨康方(低)+帕米膦酸二钠组(中+西组)：益肾骨康方浸膏剂混悬液3g/kg,1次/日×5周,灌胃,同时帕米膦酸二钠4.5mg/kg,2次/周×5周皮下注射给药；模型对照组：生理盐水0.5m1,2次/周×5周皮下注射；同时1m1,1次/日×5周,灌胃。5周后经过Micro/spectCT显像和病理切片计算各组裸鼠肺腺癌的骨转移率、骨转移部位数量、各组裸鼠病理组织切片上肿瘤细胞数量/细胞总数(%)、每周称量各组裸鼠体重观察各组裸鼠体重下降情况。
[结果]
1、低剂量“益肾骨康方”组与中+西药组骨转移率为0%,骨转移部位数为0个；而模型组骨转移率为100%,骨转移部位数为12个；高剂量和中剂量“益肾骨康方”组骨转移率分别50%和33%,骨转移部位数分别为4个和2个；帕米磷酸二钠组骨转移率为17%,骨转移部位数为1个。
2、病理组织学上可以看出,模型组裸鼠骨组织被肿瘤组织破坏严重,骨髓腔几乎完全被肿瘤细胞所填充,并可见多个破骨样细胞(多核巨细胞),骨小梁被破坏严重,部分区域可见实性肿瘤细胞团,骨小梁结构已经消失；而在骨髓腔的边缘部分,肿瘤细胞多呈活跃状态,肿瘤向外生长,骨小梁破坏严重,骨皮质广泛受侵,变薄或大片缺损；而益肾骨康方中、大剂量组虽然也发生了一定比例的骨转移,但是骨质被肿瘤细胞破坏的程度较轻,骨髓腔内可见一定的肿瘤细胞,骨小梁破坏及骨皮质受损较模型组明显减轻,骨皮质较完整。肿瘤细胞与总细胞的比值模型组明显高于“益肾骨康方”组,即模型组骨转移灶内的肿瘤细胞的数量明显高于“益肾骨康方”组(P<0.05)。
3、模型组裸鼠体重下降率也明显高于低剂量“益肾骨康方”组(P<0.05)。
[结论]
1、成功建立了肺腺癌裸鼠溶骨性骨转移模型；
2、“益肾骨康方”能够降低裸鼠肺腺癌骨转移率及骨转移部位数；
3、“益肾骨康方”能够降低裸鼠肺腺癌骨转移灶内肿瘤细胞与总细胞的比例减轻骨转移灶骨质的破坏程度；
4、“益肾骨康方”能够改善肺腺癌骨转移裸鼠的全身状况抑制其体重的下降。
第三部分：益肾骨康方对溶骨性骨转移癌FAP信号转导机制的调控作用
[目的]本研究在前期工作的基础上进一步探讨益肾骨康方对FAP信号转导的分子生物学机制的调控作用。
[方法]在裸鼠肺腺癌溶骨性骨转移模型的基础上,采用免疫组织化学染色的方法,分别观察各组裸鼠骨转移癌FAK、CD49以及CaN/NFAT信号转导关联靶点CaNAβ的表达以及组织趋化因子受体CXCR4的表达,并使用MIA4.0图像分析软件进行图像分析,统计每个高倍镜视野中表达的阳性物质的光密度值。
[结果]
1、模型组裸鼠发生了100%的骨转移,免疫组织化学结果显示模型组FAK蛋白及CD49蛋白过表达；而“益肾骨康方”低剂量组与中+西药组裸鼠未发生骨转移,免疫组织化学结果显示能够抑制FAK、CD49的表达(P<0.01)。因此“益肾骨康方”可能通过抑制FAK、CD49的过表达,从而抑制肺腺癌细胞的侵袭与转移,即“益肾骨康方”通过阻断FAK介导的FAP信号转导通路从而抑制肿瘤细胞的脱落和转移进而达到抑制肿瘤转移的作用；
2、模型组CXCR4高表达,而“益肾骨康方”低剂量组、中+西药组CXCR4(?)表达(P<0.01),表明“益肾骨康方”可能通过抑制CXCR4的表达抑制骨组织对肿瘤细胞的趋化作用,从而抑制肿瘤骨转移的发生；
3、模型组CaNAβ高表达,而“益肾骨康方”低剂量组、中+西药组以及帕米磷酸二钠组CaNAβp(?)表达(P<0.05),表明“益肾骨康方”可能通过抑制iJCaNAβ的表达从而抑制肿瘤组织对骨质的破坏,去除了癌细胞的“定居”和扩展的基础。
[结论]
1、“益肾骨康方”可能通过抑制FAK、CD49的过表达从而抑制肿瘤细胞的脱落和转移进而达到抑制肿瘤转移的作用；
2、“益肾骨康方”可能通过抑制CXCR4的表达抑制骨组织对肿瘤细胞的趋化作用,从而抑制肿瘤骨转移的发生；
3、“益肾骨康方”可能通过抑制CaNAβ的表达从而抑制肿瘤组织对骨质的溶骨性破坏。
第四部分：益肾骨康方对骨转移癌疼痛大鼠脊髓背角GFAP、c-fos、P物质蛋白表达的调控作用
[目的]研究益肾骨康方对骨转移癌疼痛大鼠脊髓背角GFAP、c-fos、P物质蛋白表达的调控作用。
[方法]在裸鼠肺腺癌溶骨性骨转移模型的基础上,采用免疫组织化学染色的方法,分别观察各组裸鼠脊髓背角GFAP、c-fos、P物质等蛋白的表达,并使用MIA4.0图像分析软件进行图像分析,统计每个高倍镜视野中表达的阳性物质的光密度值。
[结果]
1、模型组裸鼠发生了100%的骨转移,免疫组织化学结果显示模型组脊髓背角GFAP、c-fos、P物质蛋白过表达；而“益肾骨康方”低剂量组与中+西药组裸鼠未发生骨转移,免疫组织化学结果显示GFAP、c-fos、P物质蛋白的表达处于较低的水平(P<0.01)；“益肾骨康方”中、高剂量组裸鼠也发生了骨转移,但是这两组裸鼠脊髓背角GFAP、c-fos、P物质蛋白等与疼痛相关的蛋白表达明显低于模型组(P<0.05),因此“益肾骨康方”可能通过抑制裸鼠脊髓背角GFAP、c-fos、P物质等蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移所引起的疼痛,即“益肾骨康方”通过抑制裸鼠脊髓背角GFAP、c-fos、P物质蛋白的表达而起到一定的镇痛作用。
[结论]
1、“益肾骨康方”通过抑制裸鼠脊髓背角GFAP蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移所引起的疼痛；
2、“益肾骨康方”可能通过抑制裸鼠脊髓背角c-fos蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移所引起的疼痛；
3、“益肾骨康方”可能通过抑制裸鼠脊髓背角P物质蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移所引起的疼痛。
第五部分：从OPG/RANKL/RANK系统探讨益肾骨康方抑制肺腺癌溶骨性骨转移的机制
[目的]本研究拟从OPG/RANKL/RANK系统阐述补肾化瘀法中药“益肾骨康方”抑制肺腺癌骨转移的机制。
[方法]在裸鼠肺腺癌溶骨性骨转移模型的基础上,采用免疫组织化学染色的方法,分别观察各组裸鼠OPG、RANKL、IL-6的表达,并使用MIA4.0图像分析软件进行图像分析,统计每个高倍镜视野中表达的阳性物质的光密度值。
[结果]
1、模型组裸鼠发生了100%的骨转移,免疫组织化学结果显示模型组OPG蛋白低表达；而“益肾骨康方”低剂量组与中+西药组裸鼠未发生骨转移,免疫组织化学结果显示能够增加OPG蛋白的表达(P<0.01),“益肾骨康方”中、高剂量组裸鼠也发生了骨转移,但是这两组裸鼠OPG蛋白表达明显高于模型组(P<0.05)。因此“益肾骨康方”可能通过增加OPG蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移。
2、模型组裸鼠RANKL、IL-6蛋白过表达；而“益肾骨康方”低剂量组与中+西药组裸鼠未发生骨转移,与模型组相比,能够降低RANKL、IL-6蛋白的表达,(P<0.01),“益肾骨康方”中、高剂量组裸鼠也发生了骨转移,但是这两组裸鼠RANKL、IL-6蛋白的表达低于模型组(P<0.05)。因此“益肾骨康方”可能通过降低RANKL、IL-6蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移。
[结论]
1、“益肾骨康方”可能通过增加OPG蛋白的表达,从而抑制破骨细胞活性,抑制肺腺癌细胞的骨转移；
2、“益肾骨康方”可能通过抑制RNAKL、IL-6的表达从而抑制肿瘤组织对骨质的溶骨性破坏。
[创新点]
1、按照临床试验设计规范,采用平行、随机、对照临床研究原则,系统观察补肾化瘀法辨证治疗对于骨转移癌疼痛患者疼痛的治疗作用并进行疗效评价。
2、本研究采用了获得专利的CPA YANG1BM细胞系,在杨顺芳教授的指导下,采用左心室注射法成功建立了肺腺癌骨转移裸鼠模型。
3、在前期工作的基础上进一步探讨补肾化瘀法“益肾骨康方”对FAP信号转导的分子生物学机制的调控作用。
4、在前期工作的基础上进一步探讨补肾化瘀法“益肾骨康方”对骨转移癌疼痛大鼠脊髓背角GFAP、c-fos、P物质蛋白表达的调控作用,揭示该方镇痛作用的分子生物学机制。
5、从OPG/RANKL/RANK系统阐述补肾化瘀法中药“益肾骨康方”抑制肺腺癌骨转移的机制。
Osseous metastasis tumor is a common complication of late malignant tumors. The osseous metastasis tumor can affect the bones all through the body, and most frequently in the thoracolumbar spine. Most osseous metastasis tumors are multiple lesions but just a few are single lesions. The cardinal symptom of osseous metastasis tumor is pain; if the tumor or the pathological fracture affects the spinal nerve, cauda equine or spine, the specific radiating pain, zonesthesia and paraplegia will occur. If the pain thus incurred is not treated and controlled, such symptom can develop into major complications such as the pathological fracture, functional impairment, hypercalcinemia, bone marrow function repression and the paraplegia arising from the spinal nerve compression except that the pain may affect sleep and diet. The objective to treat the osseous metastasis tumor is to reduce the symptom, and prevent relevant complications such as bone fracture, raise the quality of life and increase the lifetime. The treatment of osseous metastasis tumor is one of the difficult and hot spots in clinic treatment. Since all the treatment by western medicine may have some adverse effects and addiction, disadvantageous to the rational use of drugs, it is an important orientation to seek for the medicines with less adverse effect and without addiction for the treatment of osseous metastasis tumor and osseous metastasis pain. Chinese medical science and traditional Chinese drugs can play an important role in the treatment of the pain of osseous metastasis tumor with insignificant toxicity and side effects and better effectiveness in treatment. The strictly designed clinical randomized control study and the in-depth research on the mechanism of drug action are very important. Based on the clinical differential treatment at early stage, the clinical research on the approach of Bushenhuayu to treat the pain caused by osseous metastasis tumor was developed, and the research on the signal transmission mechanism of the osteolytic osseous metastasis tumor and the molecular biological mechanism of abirritation were extensively conducted in this study. This study consists of five parts:(1) the clinical research on the traditional Chinese drug of Bushenhuayu to treat the pain caused by osseous metastasis tumor;(2) The establishment of an osteolytic osseous metastasis model by Injection of the cells of adenocarcinoma of lung into the left cardiac ventricle in nude mice to evaluate the effect of the Prescription for Yishengukang on inhibiting the occurrence of osteolytic osseous metastasis;(3) the regulating effect of the Prescription for Yishengukang on FAP signal transduction mechanism of osteolytic osseous metastasis tumor;(4) the regulating effect of the Prescription for Yishengukang on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model of osseous metastasis tumor pain;(5) Probe into the mechanism of the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung from the prospect of OPG/RANKL/RANK system.
Part Ⅰ:the clinical research on the traditional Chinese drug of Bushenhuayu to treat the pain caused by osseous metastasis tumor
[Objective] Taking the patients suffering from severe pain caused by osseous metastasis tumor as the subjects investigated, evaluation on the definite therapeutic effect of the traditional Chinese drug of Bushenhuayu at the earlier stage was conducted.
[Methods] The patients defined by the criteria for diagnosis and suffering from severe pain caused by osseous metastasis tumor were randomly divided into the treatment group and the control group and totally51observed patients were included and26cases in Chinese drug group and25cases in control group according to the design code for clinical trial, adopting the principle of parallel, randomized and controlled clinical study, and based on differentiation standard of TCM. The patients in Chinese drug group who took orally the Prescription for Yishengukang, a traditional Chinese drug of Bushenhuayu, in addition to Oxycodone&Acetaminophen Tablets (Tylox) were compared with those in the group with pure oral administration Oxycodone&Acetaminophen Tablets (Tylox). The key indicators in respect to the scores of pain influence, physical strength status (Karnofsky performance score), pain score (NRS performance score) and the dosage of Oxycodone&Acetaminophen Tablets (Tylox) were compared between the groups before and after treatment; and the therapeutic action of differential treatment by Bushenhuayu on the pain caused by osseous metastasis tumor were comprehensively analyzed, scientifically evaluated and observed.
[Results]
1. A baseline analysis was conducted on the ages of all the cases in the groups as well as the proportion of men to women in the groups to find out a proportion of46.15%and53.85%between male patients and female patients in Chinese drug group, and a proportion of48%and52%between male patients and female patients in control group and χ2test results indicated no significant difference statistically in the proportion of male-female in the two groups (P>0.05). The ages between the Chinese drug group and the control group were respectively65.15±11.43and63.28±13.48, and the t test results indicated no significant difference statistically in the ages of the two groups (P>0.05)
2. Through the comparison of the effectiveness ratio (case number and percentage) of scores of pain influence among the cases in the two groups, the results indicated the effectiveness ratio of scores of pain influence in Chinese drug group and the control group was respectively65.38%and28%and the comparison of the effectiveness ratio of scores of pain influence among the cases in the two groups had significant difference after treatment (P<0.05)
3. After14days of treatment, there were9cases of significant effect,13case of effectiveness and4case of inefficacy in Chinese drug group in terms of KRS score with a effective rate of84.61%while4cases of significant effect,9case of effectiveness and12case of inefficacy in control group with a effective rate of52.0%. through rank-sum test and after14days of treatment, the effective rate of life quality of the patients in treatment group was higher than that in control group with a statistic difference (P<0.05). The KPS score of the Chinese drug group after treatment was66.92±12.89,with a extremely significant difference in statistic compared to52.31±13.66of the Chinese drug group before treatment (P<0.01). the KPS score of the control group after treatment was59.20±13.52, with no significant difference in statistic compared to56.40±11.50of the control group before treatment (P>0.05)
4. In terms of comparison of NRS score between two groups before treatment, the Chinese drug group was5.08±1.62while that of the control group was5.12±1.59with no significant difference in statistic (P>0.05), indicating that the two groups were comparable; NRS score between two groups after treatment was slightly decreased, the Chinese drug group was2.73±1.51with a significant difference (P<0.05) compared with5.08±1.62before treatment, while the control group was4.16±2.13with no significant difference compared with5.12±1.59before treatment (P>0.05); the comparison between two groups after treatment had a significant difference (P<0.05).
5. Single day dosage of Tylox on the first day of two groups before treatment:the Chinese drug group and the control group respectively were6.04±1.82and6.56±2.31with no significant difference (P>0.05), indicating that the two groups were comparable; the single dosage of Tylox of two groups after treatment was slightly decreased, the single dosage of Tylox of the Chinese drug group was4.35±0.63with a significant difference in statistic(P<0.05) compared with that before treatment, while the single dosage of Tylox of the control group was5.60±2.06with no significant difference compared with that before treatment (P>0.05); the comparison between two groups after treatment had a significant difference (P<0.05).
6. Safety evaluation:in this experiment, all the patients were given the inspection of liver function, renal function, blood routine examination and ECG before and after treatment and the statistic results showed no significant variance, indicating that the traditional Chinese drug of Bushenhuayu was safe in use without remarkable adverse effect or toxic effect on the human systems of heart, liver, kidney and hematopoiesis. During the experiment, no adverse reaction was found in the treatment group, indicating the traditional Chinese drug of Bushenhuayu was safe in use without remarkable adverse effect or toxic effect.
[Conclusion]
1. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can increase the effective rate of pain influence score;
2. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can increase the survival quality of the patients;
3. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can decrease the NRS score and reduce pain level;
4. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can decrease the single day dosage of Lylox;
5. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu is safe and has no remarkable adverse reaction or remarkable adverse effect or toxic effect.
Part II:The establishment of an osteolytic osseous metastasis model by injection of the cells of adenocarcinoma of lung into the left cardiac ventricle in nude mice to evaluate the effect of the Prescription for Yishengukang on inhibiting the occurrence of osteolytic osseous metastasis
[Objective] An osteolytic osseous metastasis model was established in this experiment by the patented adenocarcinoma of lung cell line featured by osteolytic osseous metastasis through the method of injection into the left cardiac ventricle in nude mice, based on which the intervention effect of traditional Chinese drug was observed.
[Method] The patented CPA YANG IBM cell line was used in this research to establish an osteolytic osseous metastasis model in nude mice through the method of injection into the left cardiac ventricle under the guidance of Prof. Yang Shunfang, and the occurrence of100%osseous metastasis tumor in the model groups was eventually validated by Micro SPECT/CT tomography and pathological section, which confirmed that models was successfully made. The nude mice were randomly divided into six groups with six in each group and pharmacological intervention was given on the third day when model was made. The group with lower dosage of Prescription for Yishengukang:suspension of Yishengukang Extract3g/kg, once per day x five weeks, by intragastric administration; The group with moderate dosage of Prescription for Yishengukang:suspension of Yishengukang Extract12g/kg, once per day x five weeks, by intragastric administration; The group with higher dosage of Prescription for Yishengukang:suspension of Yishengukang Extract24g/kg, once per day x five weeks, by intragastric administration; the group with Pamidronate Disodium:Pamidronate Disodium4.5mg/kg, twice per week x five weeks, by subcutaneous injection administration; group with lower dosage of Prescription for Yishengukang plus Pamidronate Disodium (Chinese drug plus western drug group): suspension of Yishengukang Extract3g/kg, once per day x five weeks, by intragastric administration, and simultaneously Pamidronate Disodium4.5mg/kg, twice per week x five weeks, by subcutaneous injection administration; model control group:NS0.5ml, twice per week x five weeks, by subcutaneous injection administration, and simultaneously NS1ml, once per day x five weeks, by subcutaneous injection administration. After5weeks, the osseous metastasis rate and the location number of adenocarcinoma of lung in the nude mice in each group, the tumor cell number/total number of cells (%) on pathological tissue section in the nude mice in each group; and the body weight of the nude mice in each group was weighed in each week to observe the decrease of body weight in each group.
[Results]
1. The osseous metastasis rate of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group was zero and the location number of osseous metastasis was zero; while the osseous metastasis rate of the model group was100%and the location number of osseous metastasis was12; the osseous metastasis rate of the group with moderate and higher dosage of Prescription for Yishengukang was respectively50%and33%and the location number of osseous metastasis was respectively4and2; the osseous metastasis rate of the group with Pamidronate Disodium was17%and the location number of osseous metastasis was1.
2. It was observed by histopathology that the osseous tissue in the nude mice in the model group was severely destroyed by tumor tissue and the marrow cavity was almost filled out by tumor cells and several OLCs (Multinuclear Giant Cells) were observed, the bone trabecula was severely destroyed, the solid tumor cell clusters was seen in some areas and the structure of bone trabecula was disappeared; at the edge of marrow cavity, most of the tumor cells were in active status and the tumor grew out resulting in severe destroy of bone trabecula and extensive invasion, attenuation and defection of cortical bone; as for Prescription for Yishengukang, though a certain proportion of osseous metastasis occurred in the group with higher dosage, the extent for the bone mass to be destroyed by tumor cells was minor; a certain tumor cells were seen in marrow cavity but the destroy to bone trabecula and the damage to cortical bone were obviously minor compared with that of the model group and the cortical bone was relatively intact. The ratio between tumor cells and total cells of the model group was obviously higher than that of the group with Prescription for Yishengukang, namely, the quantity of tumor cell inside the osseous metastasis focus of the model group was obviously higher than that of the group with Prescription for Yishengukang (P<0.05).
3. The body weight decrease rate of the nude mice model group was obviously higher than that of the group with lower dosage of Prescription for Yishengukang (P<0.05).
The above results indicated with respect to a whole and at the cellular level that Prescription for Yishengukang had a certain inhibition on the osseous metastasis of adenocarcinoma of lung in nude mice.
[Conclusion]
1. An osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice as successfully established;
2. Prescription for Yishengukang could decrease the osseous metastasis rate and the number of osseous metastasis location of adenocarcinoma of lung in nude mice;
3. Prescription for Yishengukang could decrease the ratio between the tumor cells and the total cells inside the osseous metastasis focus of adenocarcinoma of lung in nude mice and could mitigate the destroy extent of the osseous metastasis focus to the bone mass;
4. Prescription for Yishengukang could decrease e concentration of alkaline phosphatase in the serum of nude mice with osseous metastasis of adenocarcinoma of lung;
5. Prescription for Yishengukang could improve the whole body condition of the nude mice with osseous metastasis of adenocarcinoma of lung and could inhibit their body weight decrease.
Part III:The regulating effect of the Prescription for Yishengukang on FAP signal transduction mechanism of osteolytic osseous metastasis tumor
[Objective] Further probe into the regulating effect of the Prescription for Yishengukang on the molecular biology mechanism for FAP signal transduction was conducted in this research based on the work finished at earlier stages.
[Method] On the basis of osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice, the method of Immunohistochemical staining was adopted to respectively observe the expression of CaNAβ,a target associating with the signal transduction of FAK, CD49and CaN/NFAT at the junction between osseous metastasis tumor and the bones as well as the osseous metastasis tumor in nude mice in each group and the expression of chemokine receptor CXCR4. Image analysis was conducted by image analysis software MIA4.0, and five visual fields were chosen from each section to count the optical density value of the positive substances expressed in each high magnification visual field under400Microscope Magnification.
[Results]
1.100%osseous metastasis occurred in the nude mice in model group and the immunohistochemistry data indicated that the FAK and CD49protein in the model group was over expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, the immunohistochemistry data indicated that the expression of FAK and CD49could be inhibited (P<0.01), therefore, Prescription for Yishengukang could inhibit the invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the over expression of FAK and CD49, namely, Prescription for Yishengukang could inhibit the detachment and metastasis of tumor cells by stopping the FAP signal transduction pathway mediated by FAK to actualize the effect on inhibition of tumor metastasis;
2. High expression of CXCR4in the model group vs. low expression of CXCR4(P <0.01) in the group with lower dosage of Prescription for Yishengukang, the Chinese drug plus western drug group and the group with Pamidronate Disodium indicated that Prescription for Yishengukang could inhibit the chemotaxis effect of bony tissue on the tumor cells by inhibiting the expression of CXCR4and could inhibit the occurrence of osseous metastasis of tumor;
3. High expression of CaNAβ in the model group vs. low expression of CaNAβ (P<0.01) in the group with lower dosage of Prescription for Yishengukang, the Chinese drug plus western drug group and the group with Pamidronate Disodium indicated that Prescription for Yishengukang could inhibit the destroy of tumor tissue to the bone mass by inhibiting the expression of CaNAβ, namely inhibiting the osteolytic bone resorption caused by osteoclast activation and could inhibit the osteolytic damage to bone mass in local areas and eliminate the foundation for tumor cell to "settle down" and expand.
[Conclusion]
1. The Prescription for Yishengukang could inhibit the invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the over expression of FAK and CD49, namely, Prescription for Yishengukang could inhibit the detachment and metastasis of tumor cells by stopping the FAP signal transduction pathway mediated by FAK to actualize the effect on inhibition of tumor metastasis;
2. The Prescription for Yishengukang could inhibit the chemotaxis effect of bony tissue on the tumor cells by inhibiting the expression of CXCR4and could inhibit the occurrence of osseous metastasis of tumor;3. Prescription for Yishengukang could inhibit the osteolytic damage to bone mass by inhibiting the expression of CaNAβ.
Part Ⅳ:The regulating effect of the Prescription for Yishengukang on the protein expression in dorsal horn of spinal cord in a rat model of osseous metastasis tumor pain
[Objective] The regulating effect of the Prescription for Yishengukang on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model was researched.
[Method] On the basis of osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice, the method of Immunohistochemical staining was adopted to respectively observe the expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model in each group. Image analysis was conducted by image analysis software MIA4.0, and five visual fields were chosen from each section to count the optical density value of the positive substances expressed in each high magnification visual field under400Microscope Magnification.
[Results]
1.100%osseous metastasis occurred in the nude mice in model group and the immunohistochemistry data indicated that GFAP, c-fos and P substances in dorsal horn of spinal cord in the model group were over expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, the immunohistochemistry data indicated that the expression of the protein of GFAP, c-fos and P substances was at a lower level (P<0.01); osseous metastasis occurred in the nude mice of the groups with moderate and higher dosage of Prescription for Yishengukang, but the expression of the proteins in relation to pain such as GFAP, c-fos and P substances in dorsal horn of spinal cord in nude mice in these two groups was obviously lower than that of the model group (P<0.05); therefore, Prescription for Yishengukang could inhibit the pain caused by the invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in nude mice, namely, Prescription for Yishengukang had certain abirritation by inhibiting the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in nude mice.
[Conclusion]
1. The Prescription for Yishengukang could inhibit the pain caused by the osseous invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of GFAP in dorsal horn of spinal cord in nude mice;
2. The Prescription for Yishengukang could inhibit the pain caused by the osseous invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of c-fos in dorsal horn of spinal cord in nude mice;
3. The Prescription for Yishengukang could inhibit the pain caused by the osseous invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of P substance in dorsal horn of spinal cord in nude mice.
Part V:Probe into the mechanism of the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung from the prospect of OPG/RANKL/RANK system
(Objective] The mechanism of the traditional Chinese drug, the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung was planned to be expatiated from the prospect of OPG/RANKL/RANK system.
[Method] On the basis of osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice, the method of Immunohistochemical staining was adopted to respectively observe the expression of OPG (protein expressions of osteopro tegerin), RANKL and IL-6in osseous metastasis tumor and at the junction between osseous metastasis tumor and the bones in nude mice in each group. Image analysis was conducted by image analysis software MIA4.0, and five visual fields were chosen from each section to count the optical density value of the positive substances expressed in each high magnification visual field under400Microscope Magnification.
[Results]
1.100%osseous metastasis occurred in the nude mice in model group and the immunohistochemistry data indicated that the OPG protein in the model group were down-regulated expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, the immunohistochemistry data indicated that the expression of the OPG protein could be increased (P<0.01); osseous metastasis occurred in the nude mice of the groups with moderate and higher dosage of Prescription for Yishengukang, but the expression of the OPG protein in nude mice in these two groups was obviously higher than that of the model group (P<0.05); therefore, Prescription for Yishengukang could inhibit the osseous invasion and metastasis of adenocarcinoma of lung cells through increasing the expression of OPG protein.
2. The proteins of RANKL and IL-6in nude mice in the model group were over expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, where, compared with the model group, the expression of proteins of RANKL and IL-6could be down-regulated (P<0.01), osseous metastasis occurred in the nude mice of the groups with moderate and higher dosage of Prescription for Yishengukang, but the expression of the proteins of RANKL and IL-6in nude mice in these two groups was lower than that of the model group (P<0.05); therefore, Prescription for Yishengukang could inhibit the osseous invasion and metastasis of adenocarcinoma of lung cells through decreasing the expression of proteins of RANKL and IL-6.
[Conclusion]
1. The Prescription for Yishengukang could inhibit the osseous metastasis of adenocarcinoma of lung cells through increasing OPG protein expression;
2. The Prescription for Yishengukang could inhibit the osteolytic damage of tumor tissue to the bone mass through inhibiting the expression of proteins of RANKL and IL-6.
[Innovation points]
1. The therapeutic action by differential Treatment on the pain suffered from osseous metastasis tumor was systematically observed and the curative effect was evaluated according to the design code for clinical trial, adopting the principle of parallel, randomized and controlled clinical study.
2. The patented CPA YANG1BM cell line was used in this research to establish an osteolytic osseous metastasis model in nude mice successfully through the method of injection into the left cardiac ventricle under the guidance of Prof. Yang Shunfang.
3. Further probe into the regulating effect of the Prescription for Yishengukang on the molecular biology mechanism for FAP signal transduction was conducted in this research based on the work finished at earlier stages.
4. Further probe into the regulating effect of the Prescription for Yishengukang on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model for osseous metastasis tumor pain was conducted based on the work finished at earlier stages.
5. The mechanism of the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung was expatiated from the prospect of OPG/RANKL/RANK system.
第一部分：补肾化瘀中药治疗骨转移癌疼痛的临床研究
[目的]本研究以给患者造成极大痛苦的骨转移癌疼痛为研究对象,对前期研究有确切疗效的补肾化瘀中药进行疗效评价。
[方法]按照临床试验设计规范,采用平行、随机、对照临床研究原则,根据中医辨证标准将符合诊断标准的骨转移癌疼痛患者采用随机法分为治疗组和对照组,共纳入观察病例51例,中药组26例,对照组25例。中药组患者在口服泰勒宁基础上,同时口服补肾化瘀中药“益肾骨康方”,同单纯口服氨酚羟考酮泰勒宁组进行对照。通过对疼痛影响评分、体力状况、疼痛评分、泰勒宁用量等关键性指标进行组间及治疗前后比较,综合分析、科学评价,观察补肾化瘀法辨证治疗对于骨转移癌疼痛患者疼痛的治疗作用。
[结果]
1、对全部入组病例的年龄、各组男女性别比例情况进行基线分析,中药组男患者与女患者的比例分别为46.15%和53.85%,对照组男患者与女患者的比例分别为48%和52%,χ2检验结果示两组男女比例在统计学上无显著性差异(P>0.05)。中药组和对照组年龄的分别为65.15±11.43和63.28±13.48,t检验结果示两组年
龄在统计学上无显著性差异(P>0.05)。
2、通过对两组病例疼痛影响评分有效率比较(例数／百分比),结果显示中药组与对照组的疼痛影响评分有效率分别为65.38%和28%,治疗后两组病例疼痛影响评分有效率比较有显著性差异(P<0.05)。
3、经过14天的治疗,中药组KPS评分显效9例,有效13例,无效4例,有效率达84.61%,而对照组显效4例,有效9例,无效12例,有效率为52.0%。经秩和检验,在14天的治疗后,治疗组患者的生活质量有效率高于对照组,具有统计学差异(P<0.05)。中药组治疗后KPS评分为66.92±12.89与中药组治疗前52.31±13.66相比较统计学上有极显著性差异(P<0.01)。对照组治疗后KPS评分为59.20±13.52与治疗前56.40±11.50相比统计学上无显著性差异(P>0.05)。
4、治疗前两组NRS评分比较,中药组为5.08±1.62,对照组为5.12±1.59,两组在统计学上无显著性差异(P>0.05),说明两组具有可比性；治疗后两组NRS评分都有所降低,中药组为2.73±1.51与治疗前5.08±1.62比较有显著性差异(P<0.05),对照组治疗后为4.16±2.13与治疗前5.12±1.59比较无显著性差异(P>0.05)；治疗后两组之间比较,有显著性差异(P<0.05)。
5、治疗前两组首日泰勒宁单日用量,中药组与对照组分别为6.04±1.82和6.56±2.31,两者比较无显著性差异(P>0.05),说明两组具有可比性；治疗后两组泰勒宁单日用量都有所降低,治疗组治疗后泰勒宁单日用量为4.35±0.63,与治疗前比较统计学上有显著性差异(P<0.05),对照组治疗后泰勒宁单日用量5.60±2.06与治疗前比较无显著性差异(P>0.05)；治疗后两组之间比较,有显著性差异(P<0.05)。
6、安全性评价：在本试验中全部患者治疗前后均行肝功、肾功、血常规、心电图检查,治疗前后统计结果显示无明显变化,提示补肾化瘀中药使用安全,对人体心、肝、肾、造血系统无明显毒副作用。试验期间,治疗组未发现明显不良反应。提示补肾化瘀中药安全,无明显毒副作用。
[结论]
1、补肾化瘀法治疗骨转移癌疼痛能够提高对疼痛影响评分的有效率；
2、补肾化瘀法治疗骨转移癌疼痛能够提高患者的生存质量；
3、补肾化瘀法治疗骨转移癌疼痛能够降低NRS评分,减轻疼痛水平；
4、补肾化瘀法治疗骨转移癌疼痛能够减少止痛药泰勒宁的单日用量；
5、补肾化瘀法治疗骨转移癌疼痛无明显不良反应,无明显毒副作用,是安全的。
第二部分：建立左心室注射肺腺癌溶骨性骨转移裸鼠模型评价益肾骨康方抑制溶骨性骨转移发生的作用
[目的]本实验以获得专利的具有溶骨性骨转移特性的肺腺癌细胞系通过左心室注射法建立溶骨性骨转移模型,并在此模型基础上观察中药的干预作用。
[方法]本研究采用了获得专利的CPAYANGIBM细胞系,在杨顺芳教授的指导下,采用左心室注射法建立了肺腺癌骨转移裸鼠模型,最终经过Micro SPECT/CT显像和病理切片证实模型组发生了100%的骨转移癌,证实造模成功。将裸鼠随机分为6组,每组6只,并于造模后第3天起给予药物干预。益肾骨康方低剂量组：益肾骨康方浸膏剂混悬液3g/kg,1次/日×5周,灌胃；益肾骨康组中剂量组：益肾骨康方浸膏剂混悬液12g/kg,1次/日×5周,灌胃；益肾骨康组高剂量组：益肾骨康方浸膏剂混悬液24g/kg,1次/日×5周,灌胃；帕米膦酸二钠组：帕米膦酸二钠4.5mg/kg,2次/周×5周皮下注射给药；益肾骨康方(低)+帕米膦酸二钠组(中+西组)：益肾骨康方浸膏剂混悬液3g/kg,1次/日×5周,灌胃,同时帕米膦酸二钠4.5mg/kg,2次/周×5周皮下注射给药；模型对照组：生理盐水0.5m1,2次/周×5周皮下注射；同时1m1,1次/日×5周,灌胃。5周后经过Micro/spectCT显像和病理切片计算各组裸鼠肺腺癌的骨转移率、骨转移部位数量、各组裸鼠病理组织切片上肿瘤细胞数量/细胞总数(%)、每周称量各组裸鼠体重观察各组裸鼠体重下降情况。
[结果]
1、低剂量“益肾骨康方”组与中+西药组骨转移率为0%,骨转移部位数为0个；而模型组骨转移率为100%,骨转移部位数为12个；高剂量和中剂量“益肾骨康方”组骨转移率分别50%和33%,骨转移部位数分别为4个和2个；帕米磷酸二钠组骨转移率为17%,骨转移部位数为1个。
2、病理组织学上可以看出,模型组裸鼠骨组织被肿瘤组织破坏严重,骨髓腔几乎完全被肿瘤细胞所填充,并可见多个破骨样细胞(多核巨细胞),骨小梁被破坏严重,部分区域可见实性肿瘤细胞团,骨小梁结构已经消失；而在骨髓腔的边缘部分,肿瘤细胞多呈活跃状态,肿瘤向外生长,骨小梁破坏严重,骨皮质广泛受侵,变薄或大片缺损；而益肾骨康方中、大剂量组虽然也发生了一定比例的骨转移,但是骨质被肿瘤细胞破坏的程度较轻,骨髓腔内可见一定的肿瘤细胞,骨小梁破坏及骨皮质受损较模型组明显减轻,骨皮质较完整。肿瘤细胞与总细胞的比值模型组明显高于“益肾骨康方”组,即模型组骨转移灶内的肿瘤细胞的数量明显高于“益肾骨康方”组(P<0.05)。
3、模型组裸鼠体重下降率也明显高于低剂量“益肾骨康方”组(P<0.05)。
[结论]
1、成功建立了肺腺癌裸鼠溶骨性骨转移模型；
2、“益肾骨康方”能够降低裸鼠肺腺癌骨转移率及骨转移部位数；
3、“益肾骨康方”能够降低裸鼠肺腺癌骨转移灶内肿瘤细胞与总细胞的比例减轻骨转移灶骨质的破坏程度；
4、“益肾骨康方”能够改善肺腺癌骨转移裸鼠的全身状况抑制其体重的下降。
第三部分：益肾骨康方对溶骨性骨转移癌FAP信号转导机制的调控作用
[目的]本研究在前期工作的基础上进一步探讨益肾骨康方对FAP信号转导的分子生物学机制的调控作用。
[方法]在裸鼠肺腺癌溶骨性骨转移模型的基础上,采用免疫组织化学染色的方法,分别观察各组裸鼠骨转移癌FAK、CD49以及CaN/NFAT信号转导关联靶点CaNAβ的表达以及组织趋化因子受体CXCR4的表达,并使用MIA4.0图像分析软件进行图像分析,统计每个高倍镜视野中表达的阳性物质的光密度值。
[结果]
1、模型组裸鼠发生了100%的骨转移,免疫组织化学结果显示模型组FAK蛋白及CD49蛋白过表达；而“益肾骨康方”低剂量组与中+西药组裸鼠未发生骨转移,免疫组织化学结果显示能够抑制FAK、CD49的表达(P<0.01)。因此“益肾骨康方”可能通过抑制FAK、CD49的过表达,从而抑制肺腺癌细胞的侵袭与转移,即“益肾骨康方”通过阻断FAK介导的FAP信号转导通路从而抑制肿瘤细胞的脱落和转移进而达到抑制肿瘤转移的作用；
2、模型组CXCR4高表达,而“益肾骨康方”低剂量组、中+西药组CXCR4(?)表达(P<0.01),表明“益肾骨康方”可能通过抑制CXCR4的表达抑制骨组织对肿瘤细胞的趋化作用,从而抑制肿瘤骨转移的发生；
3、模型组CaNAβ高表达,而“益肾骨康方”低剂量组、中+西药组以及帕米磷酸二钠组CaNAβp(?)表达(P<0.05),表明“益肾骨康方”可能通过抑制iJCaNAβ的表达从而抑制肿瘤组织对骨质的破坏,去除了癌细胞的“定居”和扩展的基础。
[结论]
1、“益肾骨康方”可能通过抑制FAK、CD49的过表达从而抑制肿瘤细胞的脱落和转移进而达到抑制肿瘤转移的作用；
2、“益肾骨康方”可能通过抑制CXCR4的表达抑制骨组织对肿瘤细胞的趋化作用,从而抑制肿瘤骨转移的发生；
3、“益肾骨康方”可能通过抑制CaNAβ的表达从而抑制肿瘤组织对骨质的溶骨性破坏。
第四部分：益肾骨康方对骨转移癌疼痛大鼠脊髓背角GFAP、c-fos、P物质蛋白表达的调控作用
[目的]研究益肾骨康方对骨转移癌疼痛大鼠脊髓背角GFAP、c-fos、P物质蛋白表达的调控作用。
[方法]在裸鼠肺腺癌溶骨性骨转移模型的基础上,采用免疫组织化学染色的方法,分别观察各组裸鼠脊髓背角GFAP、c-fos、P物质等蛋白的表达,并使用MIA4.0图像分析软件进行图像分析,统计每个高倍镜视野中表达的阳性物质的光密度值。
[结果]
1、模型组裸鼠发生了100%的骨转移,免疫组织化学结果显示模型组脊髓背角GFAP、c-fos、P物质蛋白过表达；而“益肾骨康方”低剂量组与中+西药组裸鼠未发生骨转移,免疫组织化学结果显示GFAP、c-fos、P物质蛋白的表达处于较低的水平(P<0.01)；“益肾骨康方”中、高剂量组裸鼠也发生了骨转移,但是这两组裸鼠脊髓背角GFAP、c-fos、P物质蛋白等与疼痛相关的蛋白表达明显低于模型组(P<0.05),因此“益肾骨康方”可能通过抑制裸鼠脊髓背角GFAP、c-fos、P物质等蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移所引起的疼痛,即“益肾骨康方”通过抑制裸鼠脊髓背角GFAP、c-fos、P物质蛋白的表达而起到一定的镇痛作用。
[结论]
1、“益肾骨康方”通过抑制裸鼠脊髓背角GFAP蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移所引起的疼痛；
2、“益肾骨康方”可能通过抑制裸鼠脊髓背角c-fos蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移所引起的疼痛；
3、“益肾骨康方”可能通过抑制裸鼠脊髓背角P物质蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移所引起的疼痛。
第五部分：从OPG/RANKL/RANK系统探讨益肾骨康方抑制肺腺癌溶骨性骨转移的机制
[目的]本研究拟从OPG/RANKL/RANK系统阐述补肾化瘀法中药“益肾骨康方”抑制肺腺癌骨转移的机制。
[方法]在裸鼠肺腺癌溶骨性骨转移模型的基础上,采用免疫组织化学染色的方法,分别观察各组裸鼠OPG、RANKL、IL-6的表达,并使用MIA4.0图像分析软件进行图像分析,统计每个高倍镜视野中表达的阳性物质的光密度值。
[结果]
1、模型组裸鼠发生了100%的骨转移,免疫组织化学结果显示模型组OPG蛋白低表达；而“益肾骨康方”低剂量组与中+西药组裸鼠未发生骨转移,免疫组织化学结果显示能够增加OPG蛋白的表达(P<0.01),“益肾骨康方”中、高剂量组裸鼠也发生了骨转移,但是这两组裸鼠OPG蛋白表达明显高于模型组(P<0.05)。因此“益肾骨康方”可能通过增加OPG蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移。
2、模型组裸鼠RANKL、IL-6蛋白过表达；而“益肾骨康方”低剂量组与中+西药组裸鼠未发生骨转移,与模型组相比,能够降低RANKL、IL-6蛋白的表达,(P<0.01),“益肾骨康方”中、高剂量组裸鼠也发生了骨转移,但是这两组裸鼠RANKL、IL-6蛋白的表达低于模型组(P<0.05)。因此“益肾骨康方”可能通过降低RANKL、IL-6蛋白的表达,从而抑制肺腺癌细胞的骨侵袭与转移。
[结论]
1、“益肾骨康方”可能通过增加OPG蛋白的表达,从而抑制破骨细胞活性,抑制肺腺癌细胞的骨转移；
2、“益肾骨康方”可能通过抑制RNAKL、IL-6的表达从而抑制肿瘤组织对骨质的溶骨性破坏。
[创新点]
1、按照临床试验设计规范,采用平行、随机、对照临床研究原则,系统观察补肾化瘀法辨证治疗对于骨转移癌疼痛患者疼痛的治疗作用并进行疗效评价。
2、本研究采用了获得专利的CPA YANG1BM细胞系,在杨顺芳教授的指导下,采用左心室注射法成功建立了肺腺癌骨转移裸鼠模型。
3、在前期工作的基础上进一步探讨补肾化瘀法“益肾骨康方”对FAP信号转导的分子生物学机制的调控作用。
4、在前期工作的基础上进一步探讨补肾化瘀法“益肾骨康方”对骨转移癌疼痛大鼠脊髓背角GFAP、c-fos、P物质蛋白表达的调控作用,揭示该方镇痛作用的分子生物学机制。
5、从OPG/RANKL/RANK系统阐述补肾化瘀法中药“益肾骨康方”抑制肺腺癌骨转移的机制。
Osseous metastasis tumor is a common complication of late malignant tumors. The osseous metastasis tumor can affect the bones all through the body, and most frequently in the thoracolumbar spine. Most osseous metastasis tumors are multiple lesions but just a few are single lesions. The cardinal symptom of osseous metastasis tumor is pain; if the tumor or the pathological fracture affects the spinal nerve, cauda equine or spine, the specific radiating pain, zonesthesia and paraplegia will occur. If the pain thus incurred is not treated and controlled, such symptom can develop into major complications such as the pathological fracture, functional impairment, hypercalcinemia, bone marrow function repression and the paraplegia arising from the spinal nerve compression except that the pain may affect sleep and diet. The objective to treat the osseous metastasis tumor is to reduce the symptom, and prevent relevant complications such as bone fracture, raise the quality of life and increase the lifetime. The treatment of osseous metastasis tumor is one of the difficult and hot spots in clinic treatment. Since all the treatment by western medicine may have some adverse effects and addiction, disadvantageous to the rational use of drugs, it is an important orientation to seek for the medicines with less adverse effect and without addiction for the treatment of osseous metastasis tumor and osseous metastasis pain. Chinese medical science and traditional Chinese drugs can play an important role in the treatment of the pain of osseous metastasis tumor with insignificant toxicity and side effects and better effectiveness in treatment. The strictly designed clinical randomized control study and the in-depth research on the mechanism of drug action are very important. Based on the clinical differential treatment at early stage, the clinical research on the approach of Bushenhuayu to treat the pain caused by osseous metastasis tumor was developed, and the research on the signal transmission mechanism of the osteolytic osseous metastasis tumor and the molecular biological mechanism of abirritation were extensively conducted in this study. This study consists of five parts:(1) the clinical research on the traditional Chinese drug of Bushenhuayu to treat the pain caused by osseous metastasis tumor;(2) The establishment of an osteolytic osseous metastasis model by Injection of the cells of adenocarcinoma of lung into the left cardiac ventricle in nude mice to evaluate the effect of the Prescription for Yishengukang on inhibiting the occurrence of osteolytic osseous metastasis;(3) the regulating effect of the Prescription for Yishengukang on FAP signal transduction mechanism of osteolytic osseous metastasis tumor;(4) the regulating effect of the Prescription for Yishengukang on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model of osseous metastasis tumor pain;(5) Probe into the mechanism of the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung from the prospect of OPG/RANKL/RANK system.
Part Ⅰ:the clinical research on the traditional Chinese drug of Bushenhuayu to treat the pain caused by osseous metastasis tumor
[Objective] Taking the patients suffering from severe pain caused by osseous metastasis tumor as the subjects investigated, evaluation on the definite therapeutic effect of the traditional Chinese drug of Bushenhuayu at the earlier stage was conducted.
[Methods] The patients defined by the criteria for diagnosis and suffering from severe pain caused by osseous metastasis tumor were randomly divided into the treatment group and the control group and totally51observed patients were included and26cases in Chinese drug group and25cases in control group according to the design code for clinical trial, adopting the principle of parallel, randomized and controlled clinical study, and based on differentiation standard of TCM. The patients in Chinese drug group who took orally the Prescription for Yishengukang, a traditional Chinese drug of Bushenhuayu, in addition to Oxycodone&Acetaminophen Tablets (Tylox) were compared with those in the group with pure oral administration Oxycodone&Acetaminophen Tablets (Tylox). The key indicators in respect to the scores of pain influence, physical strength status (Karnofsky performance score), pain score (NRS performance score) and the dosage of Oxycodone&Acetaminophen Tablets (Tylox) were compared between the groups before and after treatment; and the therapeutic action of differential treatment by Bushenhuayu on the pain caused by osseous metastasis tumor were comprehensively analyzed, scientifically evaluated and observed.
[Results]
1. A baseline analysis was conducted on the ages of all the cases in the groups as well as the proportion of men to women in the groups to find out a proportion of46.15%and53.85%between male patients and female patients in Chinese drug group, and a proportion of48%and52%between male patients and female patients in control group and χ2test results indicated no significant difference statistically in the proportion of male-female in the two groups (P>0.05). The ages between the Chinese drug group and the control group were respectively65.15±11.43and63.28±13.48, and the t test results indicated no significant difference statistically in the ages of the two groups (P>0.05)
2. Through the comparison of the effectiveness ratio (case number and percentage) of scores of pain influence among the cases in the two groups, the results indicated the effectiveness ratio of scores of pain influence in Chinese drug group and the control group was respectively65.38%and28%and the comparison of the effectiveness ratio of scores of pain influence among the cases in the two groups had significant difference after treatment (P<0.05)
3. After14days of treatment, there were9cases of significant effect,13case of effectiveness and4case of inefficacy in Chinese drug group in terms of KRS score with a effective rate of84.61%while4cases of significant effect,9case of effectiveness and12case of inefficacy in control group with a effective rate of52.0%. through rank-sum test and after14days of treatment, the effective rate of life quality of the patients in treatment group was higher than that in control group with a statistic difference (P<0.05). The KPS score of the Chinese drug group after treatment was66.92±12.89,with a extremely significant difference in statistic compared to52.31±13.66of the Chinese drug group before treatment (P<0.01). the KPS score of the control group after treatment was59.20±13.52, with no significant difference in statistic compared to56.40±11.50of the control group before treatment (P>0.05)
4. In terms of comparison of NRS score between two groups before treatment, the Chinese drug group was5.08±1.62while that of the control group was5.12±1.59with no significant difference in statistic (P>0.05), indicating that the two groups were comparable; NRS score between two groups after treatment was slightly decreased, the Chinese drug group was2.73±1.51with a significant difference (P<0.05) compared with5.08±1.62before treatment, while the control group was4.16±2.13with no significant difference compared with5.12±1.59before treatment (P>0.05); the comparison between two groups after treatment had a significant difference (P<0.05).
5. Single day dosage of Tylox on the first day of two groups before treatment:the Chinese drug group and the control group respectively were6.04±1.82and6.56±2.31with no significant difference (P>0.05), indicating that the two groups were comparable; the single dosage of Tylox of two groups after treatment was slightly decreased, the single dosage of Tylox of the Chinese drug group was4.35±0.63with a significant difference in statistic(P<0.05) compared with that before treatment, while the single dosage of Tylox of the control group was5.60±2.06with no significant difference compared with that before treatment (P>0.05); the comparison between two groups after treatment had a significant difference (P<0.05).
6. Safety evaluation:in this experiment, all the patients were given the inspection of liver function, renal function, blood routine examination and ECG before and after treatment and the statistic results showed no significant variance, indicating that the traditional Chinese drug of Bushenhuayu was safe in use without remarkable adverse effect or toxic effect on the human systems of heart, liver, kidney and hematopoiesis. During the experiment, no adverse reaction was found in the treatment group, indicating the traditional Chinese drug of Bushenhuayu was safe in use without remarkable adverse effect or toxic effect.
[Conclusion]
1. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can increase the effective rate of pain influence score;
2. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can increase the survival quality of the patients;
3. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can decrease the NRS score and reduce pain level;
4. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can decrease the single day dosage of Lylox;
5. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu is safe and has no remarkable adverse reaction or remarkable adverse effect or toxic effect.
Part II:The establishment of an osteolytic osseous metastasis model by injection of the cells of adenocarcinoma of lung into the left cardiac ventricle in nude mice to evaluate the effect of the Prescription for Yishengukang on inhibiting the occurrence of osteolytic osseous metastasis
[Objective] An osteolytic osseous metastasis model was established in this experiment by the patented adenocarcinoma of lung cell line featured by osteolytic osseous metastasis through the method of injection into the left cardiac ventricle in nude mice, based on which the intervention effect of traditional Chinese drug was observed.
[Method] The patented CPA YANG IBM cell line was used in this research to establish an osteolytic osseous metastasis model in nude mice through the method of injection into the left cardiac ventricle under the guidance of Prof. Yang Shunfang, and the occurrence of100%osseous metastasis tumor in the model groups was eventually validated by Micro SPECT/CT tomography and pathological section, which confirmed that models was successfully made. The nude mice were randomly divided into six groups with six in each group and pharmacological intervention was given on the third day when model was made. The group with lower dosage of Prescription for Yishengukang:suspension of Yishengukang Extract3g/kg, once per day x five weeks, by intragastric administration; The group with moderate dosage of Prescription for Yishengukang:suspension of Yishengukang Extract12g/kg, once per day x five weeks, by intragastric administration; The group with higher dosage of Prescription for Yishengukang:suspension of Yishengukang Extract24g/kg, once per day x five weeks, by intragastric administration; the group with Pamidronate Disodium:Pamidronate Disodium4.5mg/kg, twice per week x five weeks, by subcutaneous injection administration; group with lower dosage of Prescription for Yishengukang plus Pamidronate Disodium (Chinese drug plus western drug group): suspension of Yishengukang Extract3g/kg, once per day x five weeks, by intragastric administration, and simultaneously Pamidronate Disodium4.5mg/kg, twice per week x five weeks, by subcutaneous injection administration; model control group:NS0.5ml, twice per week x five weeks, by subcutaneous injection administration, and simultaneously NS1ml, once per day x five weeks, by subcutaneous injection administration. After5weeks, the osseous metastasis rate and the location number of adenocarcinoma of lung in the nude mice in each group, the tumor cell number/total number of cells (%) on pathological tissue section in the nude mice in each group; and the body weight of the nude mice in each group was weighed in each week to observe the decrease of body weight in each group.
[Results]
1. The osseous metastasis rate of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group was zero and the location number of osseous metastasis was zero; while the osseous metastasis rate of the model group was100%and the location number of osseous metastasis was12; the osseous metastasis rate of the group with moderate and higher dosage of Prescription for Yishengukang was respectively50%and33%and the location number of osseous metastasis was respectively4and2; the osseous metastasis rate of the group with Pamidronate Disodium was17%and the location number of osseous metastasis was1.
2. It was observed by histopathology that the osseous tissue in the nude mice in the model group was severely destroyed by tumor tissue and the marrow cavity was almost filled out by tumor cells and several OLCs (Multinuclear Giant Cells) were observed, the bone trabecula was severely destroyed, the solid tumor cell clusters was seen in some areas and the structure of bone trabecula was disappeared; at the edge of marrow cavity, most of the tumor cells were in active status and the tumor grew out resulting in severe destroy of bone trabecula and extensive invasion, attenuation and defection of cortical bone; as for Prescription for Yishengukang, though a certain proportion of osseous metastasis occurred in the group with higher dosage, the extent for the bone mass to be destroyed by tumor cells was minor; a certain tumor cells were seen in marrow cavity but the destroy to bone trabecula and the damage to cortical bone were obviously minor compared with that of the model group and the cortical bone was relatively intact. The ratio between tumor cells and total cells of the model group was obviously higher than that of the group with Prescription for Yishengukang, namely, the quantity of tumor cell inside the osseous metastasis focus of the model group was obviously higher than that of the group with Prescription for Yishengukang (P<0.05).
3. The body weight decrease rate of the nude mice model group was obviously higher than that of the group with lower dosage of Prescription for Yishengukang (P<0.05).
The above results indicated with respect to a whole and at the cellular level that Prescription for Yishengukang had a certain inhibition on the osseous metastasis of adenocarcinoma of lung in nude mice.
[Conclusion]
1. An osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice as successfully established;
2. Prescription for Yishengukang could decrease the osseous metastasis rate and the number of osseous metastasis location of adenocarcinoma of lung in nude mice;
3. Prescription for Yishengukang could decrease the ratio between the tumor cells and the total cells inside the osseous metastasis focus of adenocarcinoma of lung in nude mice and could mitigate the destroy extent of the osseous metastasis focus to the bone mass;
4. Prescription for Yishengukang could decrease e concentration of alkaline phosphatase in the serum of nude mice with osseous metastasis of adenocarcinoma of lung;
5. Prescription for Yishengukang could improve the whole body condition of the nude mice with osseous metastasis of adenocarcinoma of lung and could inhibit their body weight decrease.
Part III:The regulating effect of the Prescription for Yishengukang on FAP signal transduction mechanism of osteolytic osseous metastasis tumor
[Objective] Further probe into the regulating effect of the Prescription for Yishengukang on the molecular biology mechanism for FAP signal transduction was conducted in this research based on the work finished at earlier stages.
[Method] On the basis of osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice, the method of Immunohistochemical staining was adopted to respectively observe the expression of CaNAβ,a target associating with the signal transduction of FAK, CD49and CaN/NFAT at the junction between osseous metastasis tumor and the bones as well as the osseous metastasis tumor in nude mice in each group and the expression of chemokine receptor CXCR4. Image analysis was conducted by image analysis software MIA4.0, and five visual fields were chosen from each section to count the optical density value of the positive substances expressed in each high magnification visual field under400Microscope Magnification.
[Results]
1.100%osseous metastasis occurred in the nude mice in model group and the immunohistochemistry data indicated that the FAK and CD49protein in the model group was over expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, the immunohistochemistry data indicated that the expression of FAK and CD49could be inhibited (P<0.01), therefore, Prescription for Yishengukang could inhibit the invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the over expression of FAK and CD49, namely, Prescription for Yishengukang could inhibit the detachment and metastasis of tumor cells by stopping the FAP signal transduction pathway mediated by FAK to actualize the effect on inhibition of tumor metastasis;
2. High expression of CXCR4in the model group vs. low expression of CXCR4(P <0.01) in the group with lower dosage of Prescription for Yishengukang, the Chinese drug plus western drug group and the group with Pamidronate Disodium indicated that Prescription for Yishengukang could inhibit the chemotaxis effect of bony tissue on the tumor cells by inhibiting the expression of CXCR4and could inhibit the occurrence of osseous metastasis of tumor;
3. High expression of CaNAβ in the model group vs. low expression of CaNAβ (P<0.01) in the group with lower dosage of Prescription for Yishengukang, the Chinese drug plus western drug group and the group with Pamidronate Disodium indicated that Prescription for Yishengukang could inhibit the destroy of tumor tissue to the bone mass by inhibiting the expression of CaNAβ, namely inhibiting the osteolytic bone resorption caused by osteoclast activation and could inhibit the osteolytic damage to bone mass in local areas and eliminate the foundation for tumor cell to "settle down" and expand.
[Conclusion]
1. The Prescription for Yishengukang could inhibit the invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the over expression of FAK and CD49, namely, Prescription for Yishengukang could inhibit the detachment and metastasis of tumor cells by stopping the FAP signal transduction pathway mediated by FAK to actualize the effect on inhibition of tumor metastasis;
2. The Prescription for Yishengukang could inhibit the chemotaxis effect of bony tissue on the tumor cells by inhibiting the expression of CXCR4and could inhibit the occurrence of osseous metastasis of tumor;3. Prescription for Yishengukang could inhibit the osteolytic damage to bone mass by inhibiting the expression of CaNAβ.
Part Ⅳ:The regulating effect of the Prescription for Yishengukang on the protein expression in dorsal horn of spinal cord in a rat model of osseous metastasis tumor pain
[Objective] The regulating effect of the Prescription for Yishengukang on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model was researched.
[Method] On the basis of osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice, the method of Immunohistochemical staining was adopted to respectively observe the expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model in each group. Image analysis was conducted by image analysis software MIA4.0, and five visual fields were chosen from each section to count the optical density value of the positive substances expressed in each high magnification visual field under400Microscope Magnification.
[Results]
1.100%osseous metastasis occurred in the nude mice in model group and the immunohistochemistry data indicated that GFAP, c-fos and P substances in dorsal horn of spinal cord in the model group were over expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, the immunohistochemistry data indicated that the expression of the protein of GFAP, c-fos and P substances was at a lower level (P<0.01); osseous metastasis occurred in the nude mice of the groups with moderate and higher dosage of Prescription for Yishengukang, but the expression of the proteins in relation to pain such as GFAP, c-fos and P substances in dorsal horn of spinal cord in nude mice in these two groups was obviously lower than that of the model group (P<0.05); therefore, Prescription for Yishengukang could inhibit the pain caused by the invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in nude mice, namely, Prescription for Yishengukang had certain abirritation by inhibiting the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in nude mice.
[Conclusion]
1. The Prescription for Yishengukang could inhibit the pain caused by the osseous invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of GFAP in dorsal horn of spinal cord in nude mice;
2. The Prescription for Yishengukang could inhibit the pain caused by the osseous invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of c-fos in dorsal horn of spinal cord in nude mice;
3. The Prescription for Yishengukang could inhibit the pain caused by the osseous invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of P substance in dorsal horn of spinal cord in nude mice.
Part V:Probe into the mechanism of the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung from the prospect of OPG/RANKL/RANK system
(Objective] The mechanism of the traditional Chinese drug, the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung was planned to be expatiated from the prospect of OPG/RANKL/RANK system.
[Method] On the basis of osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice, the method of Immunohistochemical staining was adopted to respectively observe the expression of OPG (protein expressions of osteopro tegerin), RANKL and IL-6in osseous metastasis tumor and at the junction between osseous metastasis tumor and the bones in nude mice in each group. Image analysis was conducted by image analysis software MIA4.0, and five visual fields were chosen from each section to count the optical density value of the positive substances expressed in each high magnification visual field under400Microscope Magnification.
[Results]
1.100%osseous metastasis occurred in the nude mice in model group and the immunohistochemistry data indicated that the OPG protein in the model group were down-regulated expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, the immunohistochemistry data indicated that the expression of the OPG protein could be increased (P<0.01); osseous metastasis occurred in the nude mice of the groups with moderate and higher dosage of Prescription for Yishengukang, but the expression of the OPG protein in nude mice in these two groups was obviously higher than that of the model group (P<0.05); therefore, Prescription for Yishengukang could inhibit the osseous invasion and metastasis of adenocarcinoma of lung cells through increasing the expression of OPG protein.
2. The proteins of RANKL and IL-6in nude mice in the model group were over expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, where, compared with the model group, the expression of proteins of RANKL and IL-6could be down-regulated (P<0.01), osseous metastasis occurred in the nude mice of the groups with moderate and higher dosage of Prescription for Yishengukang, but the expression of the proteins of RANKL and IL-6in nude mice in these two groups was lower than that of the model group (P<0.05); therefore, Prescription for Yishengukang could inhibit the osseous invasion and metastasis of adenocarcinoma of lung cells through decreasing the expression of proteins of RANKL and IL-6.
[Conclusion]
1. The Prescription for Yishengukang could inhibit the osseous metastasis of adenocarcinoma of lung cells through increasing OPG protein expression;
2. The Prescription for Yishengukang could inhibit the osteolytic damage of tumor tissue to the bone mass through inhibiting the expression of proteins of RANKL and IL-6.
[Innovation points]
1. The therapeutic action by differential Treatment on the pain suffered from osseous metastasis tumor was systematically observed and the curative effect was evaluated according to the design code for clinical trial, adopting the principle of parallel, randomized and controlled clinical study.
2. The patented CPA YANG1BM cell line was used in this research to establish an osteolytic osseous metastasis model in nude mice successfully through the method of injection into the left cardiac ventricle under the guidance of Prof. Yang Shunfang.
3. Further probe into the regulating effect of the Prescription for Yishengukang on the molecular biology mechanism for FAP signal transduction was conducted in this research based on the work finished at earlier stages.
4. Further probe into the regulating effect of the Prescription for Yishengukang on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model for osseous metastasis tumor pain was conducted based on the work finished at earlier stages.
5. The mechanism of the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung was expatiated from the prospect of OPG/RANKL/RANK system.
引文
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