【摘要】：背景 喉癌是頭頸部常見的惡性腫瘤之一,血瘀證是其臨床常見的中醫(yī)分型。金喉片是具有活血化瘀功效的中藥復(fù)方制劑,既往的研究發(fā)現(xiàn)金喉片能夠有效改善聲帶瘀血狀態(tài)及重建聲帶局部微循環(huán),并能夠抑制喉癌內(nèi)血管增生,對(duì)抗喉癌細(xì)胞引起的血小板聚集和癌栓形成,減少血栓對(duì)喉癌細(xì)胞的保護(hù),達(dá)到延緩癌瘤生長(zhǎng)的目的。通過(guò)研究金喉片對(duì)血瘀證模型大鼠的血液流變學(xué)和舌象的影響可以定位其改善血瘀證的作用途徑,通過(guò)研究金喉片對(duì)喉癌裸鼠移植瘤中抑癌基因PTEN和致癌基因survivin的影響可以明確其抗癌的作用機(jī)理,整合這兩方面的研究成果就可以為臨床治療血瘀型喉癌提供實(shí)驗(yàn)基礎(chǔ)。 聲帶息肉、成人型喉乳頭狀瘤和喉鱗狀細(xì)胞癌分別屬于中醫(yī)“喉喑”、“喉瘤”和“喉菌”范疇,聲帶息肉屬于喉部良性增生性病變,喉乳頭狀瘤屬于喉部良性腫瘤,也屬于喉癌前病變,而喉鱗狀細(xì)胞癌則屬于喉部惡性腫瘤,從喉部良性增生性病變逐漸過(guò)渡到喉部惡性腫瘤的機(jī)制至今未明。通過(guò)聯(lián)合檢測(cè)致癌基因Survivin和抑癌基因PTEN在上述三種疾病中的表達(dá)差異可以揭示喉鱗狀細(xì)胞從增生到癌變的變化機(jī)制,再結(jié)合前面的動(dòng)物實(shí)驗(yàn)研究就可以為金喉片治療不同階段的血瘀型喉喑病在劑量、療程和時(shí)機(jī)的選擇上提供參考依據(jù)。 目的 觀察具有活血化瘀功效的金喉片對(duì)血瘀證模型大鼠的血液流變學(xué)和舌象的影響,以及對(duì)喉癌裸鼠移植瘤中致癌基因survivin和抑癌基因PTEN的影響,以此來(lái)了解金喉片的活血化瘀作用和治療喉癌的功效；同時(shí)研究聲帶息肉、成人型喉乳頭狀瘤和喉鱗狀細(xì)胞癌三種疾病中Survivin和PTEN的變化規(guī)律,以探討喉鱗狀細(xì)胞從增生到癌變的變化機(jī)制,為金喉片治療喉喑疾病的合理應(yīng)用建立參考依據(jù)。 方法 1.將隨機(jī)分組的32只雄性SD大鼠進(jìn)行14天的血瘀證造模,按折算后的金喉片常規(guī)有效劑量的1倍、3倍和6倍分別對(duì)造模后的大鼠進(jìn)行14天的灌胃,并設(shè)立生理鹽水對(duì)照組,觀察在造模開始前、造模后及用藥后大鼠血液流變學(xué)的變化。 2.將培養(yǎng)好的喉癌細(xì)胞接種到隨機(jī)分組的32只雌雄各半裸鼠背部皮下,按折算后的金喉片常規(guī)有效劑量的1倍、3倍和6倍分別對(duì)裸鼠進(jìn)行灌胃,并設(shè)立生理鹽水對(duì)照組,觀察裸鼠背部瘤體變化。在第28天取瘤,并進(jìn)行瘤體重量和體積檢測(cè),分析瘤體切片中致癌基因survivin和抑癌基因PTEN的表達(dá)差異。 3.選取符合臨床與病理診斷的聲帶息肉18例、成人型喉乳頭狀瘤10例和喉鱗狀細(xì)胞癌18例,將每個(gè)患者的病理蠟塊標(biāo)本各連續(xù)切片三張,分別進(jìn)行HE染色、Survivin和PTEN免疫組化檢測(cè)。比較致癌基因Survivin和抑癌基因PTEN在聲帶息肉、喉乳頭狀瘤和喉鱗癌三種疾病鱗狀上皮細(xì)胞中的表達(dá)差異。 結(jié)果 1.血瘀證模型大鼠按照378mg/Kg的劑量服用金喉片后血漿粘度出現(xiàn)了非常顯著的下降(P0.01),同時(shí)伴有高密度脂蛋白的顯著升高(P0.05)和低密度脂蛋白的顯著降低(P0.05),相應(yīng)大鼠舌體的瘀暗程度減輕也最為明顯(P0.05),紅細(xì)胞壓積和全血粘度無(wú)顯著變化(P0.05) 2.按照3300mg/Kg的劑量服用金喉片的裸鼠其背部皮下的喉癌移植瘤瘤體體積最小,重量最輕。全部裸鼠背部瘤體的平均密度為1.202g/cm3。Survivin和PTEN在瘤體中的陽(yáng)性率均體現(xiàn)出高劑組中劑組低劑組空白組的規(guī)律特點(diǎn),且兩者的陽(yáng)性率呈正相關(guān)關(guān)系(P0.01) 3.聲帶息肉的Survivin和PTEN陽(yáng)性率均顯著低于喉乳頭狀瘤和喉鱗狀細(xì)胞癌(P0.05),而喉乳頭狀瘤的Survivin和PTEN陽(yáng)性率均低于喉鱗狀細(xì)胞癌,但均沒(méi)有顯著性差異(P0.05),兩種免疫組化形成聲帶息肉成人型喉乳頭狀瘤≤喉鱗狀細(xì)胞癌的表達(dá)規(guī)律。在聲帶息肉、喉乳頭狀瘤及喉鱗狀細(xì)胞癌三種疾病中,PTEN的陽(yáng)性率均顯著低于Survivin (P0.05)。Survivin和PTEN在高分化喉鱗癌中的陽(yáng)性率要顯著低于中分化喉鱗癌(P0.05)。 結(jié)論 1.常規(guī)有效劑量的金喉片能夠通過(guò)升高高密度脂蛋白和降低低密度脂蛋白來(lái)顯著降低血瘀證模型大鼠的血漿粘度及改善其舌體血瘀程度,從而達(dá)到改善血瘀證的目的。 2.6倍有效劑量的金喉片則能夠有效抑制喉癌裸鼠移植瘤的生長(zhǎng),并且降低致癌基因Survivin在癌瘤中的表達(dá),抑癌基因PTEN的陽(yáng)性表達(dá)始終低于Survivin,表現(xiàn)出一種從屬性的應(yīng)激表達(dá)模式。 3. Survivin和PTEN在喉良性增生性疾病的鱗狀細(xì)胞中部分表達(dá),而在良性和惡性腫瘤中過(guò)度表達(dá),體現(xiàn)出隨惡性程度增加而逐漸升高的表達(dá)態(tài)勢(shì),同時(shí)在聲帶息肉、喉乳頭狀瘤和喉鱗癌中前者的陽(yáng)性率均高于后者。Survivin和PTEN雖然功能相反,但作用機(jī)制不同,非對(duì)立消長(zhǎng)關(guān)系,而是主從表達(dá)關(guān)系,PTEN是出于競(jìng)爭(zhēng)目的而從屬于Survivin的表達(dá),兩者的統(tǒng)一決定了鱗狀細(xì)胞癌變的速度和惡性程度。
[Abstract]:background Laryngeal carcinoma is one of the most common malignant tumors in the head and neck, and the blood stasis syndrome is a common Chinese medicine. The gold-throat tablet is a traditional Chinese medicine compound preparation with the effects of promoting blood circulation and removing blood stasis, and the prior researches have found that the gold-throat tablet can effectively improve the blood stasis state of the vocal cords and the local microcirculation of the vocal cords, The invention has the advantages of reducing the protection of the thrombus on the laryngeal cancer cell, and achieving the purpose of delaying the growth of the cancer tumor. Objective: To study the effect of gold-throat tablet on the blood rheology and the tongue image of the model rats with blood stasis syndrome. The effect of the gold-throat tablet on the expression of the tumor suppressor gene PTEN and the oncogenic gene survivin in the transplanted tumor of the laryngeal carcinoma in nude mice can be determined by studying the effect of the gold-throat tablet on the expression of the tumor suppressor gene PTEN and the cancer-induced gene survivin in the nude mice of the laryngeal carcinoma. The results of the two aspects can provide experimental basis for clinical treatment of blood stasis type laryngeal carcinoma. The vocal cord polyp, the adult-type laryngeal papilloma and the laryngeal squamous cell carcinoma belong to the "laryngeal mask", the "Laryngeal tumor" and the "Larynaria" of the traditional Chinese medicine, Precancerous lesion, while laryngeal squamous cell carcinoma is a malignant tumor of the larynx, from the benign hyperplasia of the larynx to the mechanism of the malignant tumor of the throat. The expression of Survivin and tumor suppressor gene PTEN in the above three diseases can reveal the change mechanism of laryngeal squamous cell from proliferation to canceration. The reference is provided on the selection of the dose, course of treatment, and timing Kauai Objective: To observe the effects of gold-throat tablets with blood stasis removing and blood stasis removing on blood rheology and tongue image of rats with blood stasis syndrome, as well as the effect of survivin and tumor suppressor gene PTEN on cancer gene survivin and tumor suppressor gene PTEN in nude mice of laryngeal carcinoma, so as to understand the effects of promoting blood circulation and removing blood stasis. The changes of the expression of Survivin and PTEN in the three kinds of diseases of vocal polyp, adult type laryngeal papilloma and laryngeal squamous cell carcinoma were studied, and the mechanism of the change of laryngeal squamous cell from proliferation to canceration was discussed. to build According to the method 1,32 male SD rats of the random group were established in 14 days of blood stasis syndrome, and the rats were given a normal effective dose of 1,3 and 6 times the conventional effective dose of the golden-throat tablets, respectively, and the normal saline was established. In the group, the model was observed before and after the start of the model. 2. The cultured laryngeal carcinoma cells were inoculated to the back of 32 male and semi-nude mice, which were randomly divided, and the mice were given an effective dose of 1-,3-and 6-fold, respectively, and the normal saline was established. The tumor was taken on the 28th day and the weight and volume of the tumor were detected, and the oncogenic gene survivin and the tumor in the tumor were analyzed. 3.18 cases of vocal cord polyp,10 cases of adult type laryngeal papilloma and 18 cases of laryngeal squamous cell carcinoma were selected according to the clinical and pathological diagnosis. The expression of survivin and tumor suppressor gene PTEN in the vocal cord polyp, the laryngeal papilloma and the laryngeal squamous cell carcinoma were compared. disease of seed Results 1. The blood-stasis syndrome model rats had a very significant decrease in plasma viscosity after the administration of the gold-throat tablets at a dose of 378 mg/ Kg (P0.01), accompanied by a marked increase in high-density lipoprotein (P0.05). And the blood stasis and dark level of the corresponding rat's tongue were also the most obvious (P0.05). There was no significant change in the hematocrit and the total blood viscosity (P0.05)2. The nude mice with the gold-throat tablets were administered in a dose of 3300 mg/ Kg. The average density of the tumor in the back of the nude mice was 1.202 g/ cm3, and the positive rate of Survivin and PTEN in the tumor body was 1.202 g/ cm3. the rule of the blank group of the low-dose group of the medium-dose group in the high-dose group is reflected, The positive rate of Survivin and PTEN in vocal cord polyp was lower than that of laryngeal papilloma and laryngeal squamous cell carcinoma (P0.05), while the positive rate of Survivin and PTEN in laryngeal papilloma was lower than that of laryngeal papilloma and laryngeal squamous cell carcinoma (P0.05). There was no significant difference in laryngeal squamous cell carcinoma (P0.05). The positive rate of PTEN was lower than that of Survivin (P0.05) in the three diseases of vocal polyp, laryngeal papilloma and laryngeal squamous cell carcinoma (P <0.05). medium Conclusion 1. The conventional effective dose of gold-throat tablets can significantly lower the blood stasis syndrome model rats by increasing the high-density lipoprotein and reducing the low-density lipoprotein. The plasma viscosity and the degree of blood stasis of the tongue body are improved, so that the purpose of improving the blood stasis syndrome is achieved. The 2.6 times effective dose of the gold-throat tablet can effectively inhibit the growth of the transplanted tumor in the nude mouse of the laryngeal cancer, and reduce the expression of the cancer gene Survivin in the tumor and the positive table of the tumor suppressor gene PTEN. The expression of Survivin and PTEN in the squamous cell of the benign and malignant diseases of the larynx is always lower than that of the Survivin, and the expression of Survivin and PTEN in the squamous cell of the benign and malignant tumor of the larynx is always lower than that of the Survivin, and the expression of Survivin and PTEN in the benign and malignant tumor is excessive. The expression of the expression, which is gradually increased with the increase of the degree of malignancy. The positive rate of the former is higher in the vocal polyp, the laryngeal papilloma and the laryngeal squamous cell carcinoma, but the positive rate of the former is higher than that of the latter.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.65

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