【摘要】：實驗背景及目的 骨肉瘤(Osteosarcoma)亦稱為成骨肉瘤,是高度惡性的原發(fā)性骨腫瘤,發(fā)病年齡在10-30歲之間,發(fā)病率居原發(fā)性惡性腫瘤之首。骨肉瘤的病程進展很快,在臨床確診病例中,80%的患者已發(fā)現(xiàn)有血行遠隔轉移。經(jīng)過手術前動脈灌注化療結合術后化療,能夠讓多數(shù)患者避免截肢,明顯提高了生存質量,預后有所改善,生存率有了明顯提高。但是,骨肉瘤較高的侵襲轉移能力以及對化療藥物的耐藥,使得部分患者在接受了規(guī)范手術和化療以后,仍然出現(xiàn)早期復發(fā)或早期轉移。p27是調節(jié)細胞周期G1/S期的一種抑制因子,在維持細胞周期順利進行中起關鍵性作用。體外實驗研究發(fā)現(xiàn),作為一個細胞周期抑制因子,p27不僅能抑制正常的血管內皮細胞增殖和遷移,也能誘導細胞凋亡。實驗發(fā)現(xiàn)p27在體內能夠抑制各種原發(fā)性腫瘤和轉移性腫瘤的生長,p27基因的缺失是導致骨肉瘤的發(fā)生和病變進展的重要原因之一。根據(jù)報道,p27基因在人類骨肉瘤移植瘤裸鼠模型中,可以改善腫瘤裸鼠的預后,緩解癥狀,延長生存時間。中藥“片仔癀”具有清熱解毒,減少腫脹和軟堅散結的功效,能夠促進血液循環(huán),消除血瘀,臨床多用于消化系統(tǒng)腫瘤的治療以及防治腫瘤化療過程中的肝損害。有研究證實片仔癀對骨肉瘤移植瘤細胞具有誘導凋亡的作用。文獻報告人類骨肉瘤細胞株MG63、 Saos-2等,經(jīng)細胞培養(yǎng)技術移植于裸鼠骨髓腔內可形成骨肉瘤病理模型；而腺相關病毒可作為p27基因載體,并在動物體內表達p27蛋白,且具有相關生物活性。因此,本實驗選取人類骨肉瘤Saos-2細胞株,建立裸鼠原位移植瘤模型,使用腺相關病毒作為p27基因載體,分別觀察和比較p27基因、中藥“片仔癀”以及兩者聯(lián)合應用對人類骨肉瘤生長的抑制作用,為以后骨肉瘤的基因治療以及中藥治療提供實驗依據(jù)。 實驗材料 1、細胞、藥物及試劑：人類骨肉瘤Saos-2細胞系,購于上海超研生物科技有限公司；片仔癀(主要成分含有麝香、牛黃、三七、蛇膽等),購于漳州片仔癀制藥有限公司(生產(chǎn)批號：H.M.L.N.Z20080011);轉染raav-p27的重組腺相關病毒溶液、空載體重組腺相關病毒(rAAV-MCS)懸液,購于西安華光生物工程公司；胎牛血清(FBS),購于HyClone公司(NE, USA), p27抗體,購于上海寶特生命科學發(fā)展有限公司；酶鏈親和素-生物素(SABC)試劑盒,購于上海復中生物技術有限公司；兔抗β-actin抗體,美國Santa Cruze公司產(chǎn)品,辣根過氧化物酶標記的羊抗兔二抗,美國Santa Cruze公司產(chǎn)品；ECL試劑盒,美國Santa Cruze公司產(chǎn)品；BCA蛋白濃度測定試劑盒,美國Bio-Rad公司產(chǎn)品。 2、主要儀器設備：血紅細胞計數(shù)器(上海實驗室儀器有限公司,中國);半干轉印儀(Semidry Transfer system,美國Bio-Rad公司)；紫外凝膠自動成像儀(上海勤翔科學儀器有限公司,中國) 實驗方法 1、實驗動物： BALB/C裸鼠30只,SPF級無特殊病原體,3-4周齡,體重15-20g,雌雄不限,規(guī)格為：CAnN.Cg-Foxn1nu/Cr1VR,購自上海寶特生命科學發(fā)展有限公司。飼養(yǎng)條件：恒溫25-27℃,濕度45%-50%,半屏障系統(tǒng)環(huán)境下飼養(yǎng),食用滅菌處理過的飼料、水,高效過濾系統(tǒng)處理過的空氣,每小時換氣10-15次,一天10小時照明,14小時在無照明黑暗條件下飼養(yǎng)。 2、人類骨肉瘤Saos-2細胞株的細胞培養(yǎng)： 細胞培養(yǎng)液的制備：采用Roswell Park Memorial Institute (RPMI)-1640培養(yǎng)液,加入10%標準胎牛血清,2mmol/L-谷氨酰銨,100U/ml青霉素和0.1mg/ml鏈霉素,過濾和消毒后,4℃儲存?zhèn)溆�。將裝有人類骨肉瘤Saos-2細胞株的凍存管從液氮罐里取出,解凍、洗滌、離心處理后,將細胞懸液轉移至10ml培養(yǎng)皿中,放置在37℃,5%CO2培養(yǎng)箱中過夜。當細胞生長至相對密度80%左右時,加入胰酶消化液,在37℃,5%的CO2培養(yǎng)箱里消化2-3分鐘,然后加入2ml細胞培養(yǎng)液終止細胞消化,此時,在顯微鏡下可觀察到大多數(shù)的游離圓形細胞。用吸管將細胞充分攪勻后,靜置。離心棄上清,加入5ml細胞培養(yǎng)液,吹打均勻。用細胞計數(shù)器計數(shù)后備用。 3、在無菌條件下,用4%水合氯醛(10μ L/g)將裸鼠麻醉。然后用1ml規(guī)格注射器4號針頭吸取0.1ml細胞懸液(1×107個細胞／注射器)注射到裸鼠右側腋窩皮下,每次注射兩只裸鼠。2-4周后,待觀察到裸鼠皮下移植瘤生長至1.0cm×1.0cm×1.5cm大小后,處死裸鼠,在無菌條件下取除骨肉瘤瘤體組織。將得到的腋窩腫瘤組織處理成0.1cm×0.1cm×0.1cm大小后再移植在新的裸鼠右后腿上端脛骨骨髓腔內,然后將裸鼠放回飼養(yǎng)籠,觀察腫瘤的生長。此時,人類骨肉瘤Saos-2細胞株裸鼠原位模型建立成功。腫瘤細胞接種后,動物生長狀況良好,在接種Saos-2細胞株5天之后,可見腫瘤組織在裸鼠皮下已隨時間增長。接種六周后,沒有出現(xiàn)動物死亡,腫瘤相對體積增長率為92.7%。 4、實驗分組及處理： 腫瘤細胞接種一周后,待腫瘤生長至0.8cm時,挑選30只腫瘤體積相近的裸鼠按隨機數(shù)字表隨機分為5組,每組6只： 空白對照組,用100μLPBS注射腫瘤部位,3天1次； 空載體對照組,用100μL多克隆位點重組腺相關病毒(rAAV-MCS)懸液注射腫瘤部位,空載體病毒溶液濃度1×1011/L,3天1次； 片仔癀藥物組,在腫瘤形成后,按0.01ml/g劑量,每天早晚各喂食給藥一次,共兩次給藥。 p27基因組,用100μL raav-p27病毒溶液注射,病毒溶液濃度1×1011/L注射腫瘤部位,3天1次。 5.聯(lián)合治療組,用100μL raav-p27病毒溶液注射腫瘤部位,3天1次,同時喂食片仔癀溶液,一天早晚兩次給藥。 在裸鼠接種之后,觀察并記錄它們的精神狀態(tài),體重,食欲、糞便和尿液。如果實驗中發(fā)現(xiàn)裸鼠死亡,解剖并分析死亡原因。 腫瘤細胞接種6周之后,用頸椎脫臼法將所有裸鼠處死。剝離腫瘤組織,稱重并記錄腫瘤重量,計算腫瘤生長抑制率,計算公式為：(對照組瘤重-實驗組瘤重)／對照組瘤重×100%。在動物模型建立之后,用游標卡尺測量每組裸鼠的第1,4,8,12,16,18和22天腫瘤的最大和最小直徑,并應用Steel公式V=1/2ab2計算體積,其中a,b分別是腫瘤的最大和最小直徑。 5、免疫組織化學染色：將腫瘤組織用福爾馬林固定,石蠟包埋后進行免疫組織化學方法(IHC)染色。使用鼠抗p27抗體(一抗)及堿性磷酸酶標記的馬抗鼠IgG(二抗),運用鏈霉親和素-生物素復合物(SABC法),并運用雙重染色法檢測p27的蛋白表達。使用光學顯微鏡觀察,在放大10倍的視野內統(tǒng)計陽性細胞表達率,來判斷p27的表達情況。 6、蛋白質印跡分析：從腫瘤組織細胞中提取蛋白,然后用抗p27兔單抗和兔抗β-actin內參抗體來進行免疫印跡實驗(Western-Blot)。經(jīng)ECL化學發(fā)光底物曝光后,用實驗室圖像分析軟件計算蛋白含量。 7、統(tǒng)計分析：采用SPSS10.0分析軟件程序來進行統(tǒng)計分析。數(shù)據(jù)采用平均值±標準差(X±S),與對比組進行單向方差分析和LSD分析。 結果 1、動物模型的癥狀觀察：及腫瘤細胞移植到裸鼠體內7周之后,p27基因組,聯(lián)合治療組以及片仔癀藥物組所有的裸鼠都存活下來,空白對照組和空載體對照組各有1只死亡�？瞻讓φ战M和空載體對照組的裸鼠腫瘤體積增長非常明顯,且兩者之間沒有顯著差異,裸鼠的精神狀態(tài)和食欲下降,尿液顏色偏暗,糞便偏干,體重下降。片仔癀藥物組的裸鼠腫瘤增長相對較緩慢,精神狀態(tài)良好,食欲正常,體重變化不大,糞便和尿液的顏色和性狀均正常。p27基因組的裸鼠腫瘤增長同樣較慢,精神狀態(tài)一般,食欲略有下降,體重隨著腫瘤體積增加有所增長,但體型偏瘦,偶爾還存在腹瀉。聯(lián)合治療組的腫瘤生長抑制明顯,裸鼠的精神狀態(tài)很好,飲食、糞便和尿液正常,沒有腹瀉現(xiàn)象,但體重略有下降。 2、腫瘤生長情況和抑制率統(tǒng)計：空白對照組與空載體對照組的腫瘤體積相近(P0.05)。在第8,12,16,18和22天記錄的腫瘤體比較中,p27基因組和片仔癀藥物組與空白對照組相比,呈明顯下降(P0.05)；聯(lián)合治療組與片仔癀藥物組或者p27基因組相比,也是明顯減少(P0.05)。在腫瘤平均重量比較上,片仔癀藥物組和p27基因組比空白對照組輕(P0.05)。聯(lián)合治療組比片仔癀藥物組或者p27基因組也要輕(P0.05)。因此,可判斷在腫瘤抑制率上,片仔癀藥物組和p27基因組比空白對照組高(P0.05)。聯(lián)合治療組比片仔癀藥物組或者p27基因組同樣也高(P0.05)。 3、p27蛋白表達情況：經(jīng)染色后的p27蛋白陽性細胞,可在細胞核里觀察到棕黃色顆粒。在p27蛋白陽性率統(tǒng)計上,p27基因組比空白對照組有顯著提高(P0.05),聯(lián)合治療組比p27基因組或者片仔癀藥物組也有明顯提高(P0.05)。免疫印跡試驗顯示在p27基因表達量比較上,p27基因組比空白對照組高(P0.05),聯(lián)合治療組比p27基因組或者片仔癀高(P0.05) 結論 1、Saos-2人類骨肉瘤細胞株經(jīng)培養(yǎng)傳代后可移植裸鼠獲得腫瘤原位生長的動物模型。 2、腺相關病毒作為基因載體對腫瘤生長無影響,轉染p27基因的腺相關病毒可增加腫瘤組織內p27蛋白的表達。 3、p27基因、片仔癀均有抑制骨肉瘤生長的作用,二者聯(lián)合應用時對腫瘤生長的抑制作用明顯增強。 意義 證實了p27重組腺相關病毒可用于骨肉瘤的研究和治療,p27基因與中藥片仔癀聯(lián)合應用對骨肉瘤的抑制有協(xié)同作用。初步探討了腺相關病毒作為基因載體用于腫瘤基因治療的可能性,在骨肉瘤的臨床治療方面,為p27基因聯(lián)合中藥片仔癀的療效研究,進一步奠定了理論基礎。
[Abstract]:Experimental background and purpose
Osteosarcoma (Osteosarcoma), also known as osteosarcoma, is a highly malignant primary bone tumor with a incidence of 10-30 years of age. The incidence of osteosarcoma is the first of primary malignant tumors. The course of osteosarcoma is progressing rapidly. In clinical cases, 80% of the patients have been found to have distant metastasis. Treatment, which allows most patients to avoid amputation, significantly improves the quality of life, improves the prognosis, and has a significant improvement in survival. However, the high invasion and metastasis ability of osteosarcoma and the resistance to chemotherapeutic drugs make some patients still have early recurrence or early metastasis.P27 after receiving standardized surgery and chemotherapy. A inhibitory factor in the cell cycle G1/S stage plays a key role in maintaining a smooth cell cycle. In vitro studies have found that, as a cell cycle inhibitor, p27 can not only inhibit the proliferation and migration of normal vascular endothelial cells, but also induce apoptosis. It is found that p27 can inhibit the primary swelling in the body. The growth of tumor and metastatic tumor, the deletion of p27 gene is one of the important reasons for the occurrence and progression of osteosarcoma. According to the report, the p27 gene in the nude mice model of human osteosarcoma transplanted tumor can improve the prognosis, relieve symptoms and prolong the survival time of tumor nude mice. The effect of soft hard and loose knot can promote blood circulation, eliminate blood stasis, use the treatment of digestive system tumor and prevent the liver damage in the process of tumor chemotherapy. The pathological model of osteosarcoma can be formed in the bone marrow cavity of nude mice. Adeno-related virus can be used as a carrier of p27 gene and expression of p27 protein in animals, and it has related biological activity. Therefore, this experiment selected human osteosarcoma Saos-2 cell lines, established the orthotopic xenograft model in nude mice, and used adeno-related virus as the p27 gene carrier. The inhibitory effects of p27 gene, Chinese herbal medicine and the combination of them on the growth of human osteosarcoma were observed and compared, and the experimental basis was provided for the gene therapy of osteosarcoma and the treatment of traditional Chinese medicine.
Experimental materials
1, cells, drugs and reagents: human osteosarcoma Saos-2 cell line, purchased in Shanghai Chao Yan Biological Technology Co., Ltd. (mainly composed of musk, bezoar, 37, snake gall), purchased in Zhangzhou Zai Zai Jin Pharmaceutical Co., Ltd. (production batch number: H.M.L.N.Z20080011); recombinant adeno-associated virus solution transfected with raav-p27, unloaded body weight group Adeno-related virus (rAAV-MCS) suspension, purchased in Xi'an Huaguang bioengineering company; fetal bovine serum (FBS), purchased in HyClone (NE, USA), p27 antibody, purchased in Shanghai Life Science Development Co., Ltd., enzyme chain avidin biotin (SABC) kit, purchased in Shanghai compound Biotechnology Co., Ltd., Rabbit anti beta -actin antibody, American Santa Cruze company products, horseradish peroxidase labelled Sheep anti rabbit two resistance, American Santa Cruze company products; ECL kit, Santa Cruze company products in the United States; BCA protein concentration test kit, American Bio-Rad company product.
2, the main instrument and equipment: blood red cell counter (Shanghai Laboratory Instrument Co., Ltd., China); semi dry transfer printer (Semidry Transfer system, American Bio-Rad company); ultraviolet gel automatic imaging instrument (Shanghai Qin Xiang Science Instrument Co., Ltd., China)
Experimental method
1, experimental animals:
BALB/C nude mice 30, SPF class no special pathogens, 3-4 weeks old, weight 15-20g, male and female, the specification is: CAnN.Cg-Foxn1nu/Cr1VR, purchased from Shanghai Bao Life Science Development Co., Ltd.. Feeding conditions: constant temperature 25-27, humidity 45%-50%, half barrier system environment, food, water, efficient filtration system treated The air is ventilated 10-15 times per hour, 10 hours a day, and 14 hours under dark conditions without lighting.
2, the cell culture of human osteosarcoma Saos-2 cell line:
Preparation of cell culture solution: using Roswell Park Memorial Institute (RPMI) -1640 culture solution, adding 10% standard fetal bovine serum, 2mmol/L- glutamyl ammonium, 100U/ml penicillin and 0.1mg/ml streptomycin, after filtration and disinfection, stored at 4 C. The cryopreservation tube containing human osteosarcoma Saos-2 cell line is removed, thawing, washing, and away from the liquid nitrogen tank. After the heart treatment, the cell suspension was transferred to the 10ml culture dish and placed in the 5%CO2 culture box for the night. When the cells grew to the relative density of about 80%, the cells were added to the digestive juice, digested at 37, 5% CO2 incubator for 2-3 minutes, and then added to the 2ml cell culture to terminate the cell digestion. At this time, the majority of the cells could be observed under the microscope. Free round cells. Stir the cells thoroughly with a straw and set aside. Discard the supernatant by centrifugation, add 5 ml cell culture medium and blow evenly. Count the cells with a cell counter and reserve them.
3, under aseptic conditions, the nude mice were anesthetized with 4% chloral chloral (10 L/g). Then the 0.1ml cell suspension (1 x 107 cells / syringes) was injected into the right armpit of the nude mice with the 1ml specification syringe needle 4 needle. After each injection of two nude mice for.2-4 weeks, it was observed that the subcutaneous transplanted tumor of the nude mice was grown to 1.0cm * 1.0cm x 1.5cm, and was executed. In nude mice, the osteosarcoma tissue was removed under the aseptic condition. After the axillary tumor tissue was treated as 0.1cm x 0.1cm x 0.1cm, the bone marrow cavity of the right hind leg of the nude mice was retransplanted into the bone marrow cavity of the right hind leg of the nude mice. Then the nude mice were put back in the cage to observe the growth of the tumor. At this time, the human osteosarcoma Saos-2 cell line in nude mice was established successfully. After inoculation of the tumor cells, the animals grew well. After 5 days of inoculation of the Saos-2 cell line, the tumor tissue had been growing subcutaneously in the nude mice. After six weeks of inoculation, no animal death was found, and the relative volume growth rate of the tumor was 92.7%.
4, experimental grouping and processing:
Thirty nude mice with similar tumor volume were randomly divided into five groups at the time of tumor growth of 0.8 cm after one week of inoculation.
In blank control group, tumor sites were injected with 100 micron LPBS for 1 days in 3 days.
In empty vector control group, the tumor site was injected with 100 mu L polyclonal site recombinant adeno-associated virus (rAAV-MCS) suspension. The concentration of empty vector virus solution was 1 *1011/L, once every 3 days.
After the tumor was formed, the drug group was given 0.01ml/g dose once a day, two times a day.
The p27 genome was injected with 100 L raav-p27 virus solution. The concentration of virus solution was 1 x 1011/L injection site, 3 days 1 times.
5. In the combined treatment group, the tumor site was injected with 100 mu L raav-p27 virus solution once every 3 days, and the tablets were fed with gall solution twice a day.
After inoculation in the nude mice, the mental state, weight, appetite, feces and urine were observed and recorded. The death of nude mice was found in the experiment and the cause of death was dissected and analyzed.
After 6 weeks of inoculation of tumor cells, all nude mice were killed by dislocated cervical vertebra. The tumor tissue was stripped, weighed and recorded, and the tumor growth inhibition rate was recorded. The formula was as follows: (control group tumor weight experimental group tumor weight) / control group of tumor weight x 100%. was established by using vernier caliper to measure the 1,4,8,12,16 of nude mice in each group. On the 18th and 22nd days, the maximum and minimum diameters of the tumor were calculated by Steel's formula V=1/2ab2, where a and B were the maximum and minimum diameters of the tumor, respectively.
5, immunohistochemical staining: the tumor tissue was immobilized with formalin, and the paraffin was embedded in the immunohistochemical staining method (IHC). The IgG (two anti) of the horse anti rat rat was labeled with anti p27 antibody (one antibody) and alkaline phosphatase, and the streptomycin biotin complex (SABC method) was used. The protein expression of p27 was detected by double staining. The expression of p27 was measured by optical microscopy, and the expression of positive cells was counted in a 10 fold field of view.
6, Western blot analysis: extract protein from tumor tissue cells, and then use anti p27 rabbit monoclonal antibody and Rabbit anti beta -actin internal reference antibody to carry out immunoblotting test (Western-Blot). After exposure to ECL chemiluminescence substrate, the protein content was calculated by laboratory image analysis software.
7, statistical analysis: the SPSS10.0 analysis software program was used to carry out statistical analysis. The data used mean mean standard deviation (X + S), and the one-way ANOVA and LSD analysis were carried out with the contrast group.
Result
1, the observation of the animal model: after the tumor cells were transplanted into the nude mice for 7 weeks, the p27 genome, the combined treatment group and all the nude mice were alive, with 1 deaths in the blank control group and the empty carrier control group. The growth of the tumor volume in the blank control group and the empty carrier group was very obvious, and two There was no significant difference in the mental state and appetite of the nude mice, the dark color of the urine, the dry feces and the loss of weight. The tumor growth of the nude mice was relatively slow, the mental state was good, the appetite was normal, the body weight changed little, the color of the feces and urine and the characters of the nude mice in the normal.P27 genome were the same. Slow, general mental state, a slight decline in appetite, weight increase with the increase of tumor volume, but the body is thin and occasional diarrhea. The tumor growth inhibition in the combined treatment group is obvious, the mental state of the nude mice is very good, the diet, feces and urine are normal, there is no diarrhea phenomenon, but the weight has a slight decline.
2, the tumor growth and inhibition rate: the volume of tumor in the blank control group and the empty vector control group was similar (P0.05). In the comparison of the tumor bodies recorded on the 8,12,16,18 and the 22 days, the p27 genome and the tablet group were significantly lower than those in the blank control group (P0.05). In comparison with the average weight of the tumor (P0.05), the drug group and the p27 genome were lighter than those in the blank control group (P0.05). The combined treatment group was lighter than that of the drug group or the p27 genome (P0.05). Therefore, in the tumor inhibition rate, the drug group and the p27 genome were higher than the blank control group (P0.05). The combined treatment group was also higher than that of pizin medicine group or p27 genome (P0.05).
3, p27 protein expression: after dyed p27 protein positive cells, the brown yellow granules can be observed in the nucleus. In the positive rate of p27 protein, the p27 genome is significantly higher than that in the blank control group (P0.05). The combined treatment group is also significantly higher than the p27 genome or the tablet group (P0.05). The Western blot test shows that in P2, the immunoblotting test shows P2 Compared with the blank control group, p27 genome was higher (P 0.05), and the combined treatment group was higher than p27 genome or litter gall (P 0.05).
conclusion
1, Saos-2 human osteosarcoma cell line can be transplanted into nude mice to obtain an animal model of tumor growth in situ.
2. Adeno-associated virus as a gene vector has no effect on tumor growth. Adeno-associated virus transfected with p27 gene can increase the expression of p27 protein in tumor tissue.
3. Both p27 gene and Tablet gall can inhibit the growth of osteosarcoma, and the inhibitory effect on the growth of osteosarcoma is obviously enhanced when they are combined.
Significance
The recombinant adeno-associated virus (p27) can be used in the study and treatment of osteosarcoma, and the combination of p27 gene and traditional Chinese medicine has synergistic effect on the inhibition of osteosarcoma. The possibility of adeno-related virus as a gene carrier for tumor gene therapy is preliminarily discussed. In the treatment of osteosarcoma, the p27 gene is combined with Chinese herbal medicine. The research of curative effect has laid the theoretical foundation further.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R738.1

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