小干擾核糖核酸治療神經外科疾病---以神經病變痛及神經腫瘤為例

Small interfering RNAs for treatment of neurosurgical diseases, specifically involving neuropathic pain and neural tumors

蔡明達(Ming-Dar Tsai)

指導教授 ： 謝松蒼 ； 孫維仁

國立臺灣大學/醫學院/臨床醫學研究所/博士(2016年)

https://doi.org/10.6342/NTU201610407

摘要

本篇論文研究分為三部分，第一部分是以緩激肽B2受體小干擾核糖核酸(Bradykinin B2 receptor siRNA)在大白鼠背根神經節阻斷疼痛傳導而達到鎮痛效果。第二部分是以端粒酶小干擾核糖核酸(telomerase siRNA)引導神經母細胞瘤癌細胞(neuroblastoma cell)產生核凝結及細胞週期停動；第三部分是以棉酚(Gossypol)引導經由Smac, p53及caspase途徑的視網膜母細胞腫瘤細胞凋亡。
小干擾核糖核酸(small interfering RNA; siRNA)是長度20-25個核甘酸的雙股RNA，主要參與RNA干擾(RNAi)現象，以帶有專一性的方式調節基因的表達。小干擾RNA可經由多種不同轉染(transfection)技術導入細胞內，並對特定基因產生具專一性的基因表現減量(knock-out)效果，已經成為研究基因功能與開發新藥物目標的一項重要工具。
第一部分研究以大白鼠神經病變痛動物模式進行，將大白鼠麻醉後，左側腰椎第六條神經使用外科手術用鈦金屬夾夾住，造成神經損傷，因此就成為神經病變痛動物模式。大白鼠的接受手術後，在不同的足底觸覺刺激程度時觀察牠左腳收起時所需的時間，用來作為對疼痛感覺敏銳度的評估。在對照組是將神經夾起，實驗組則將神經夾起同時系列的注射緩激肽B2受體小干擾核糖核酸，實驗動物觀察到2周的時間，同時重複性的比較大白鼠雙下肢對於觸感刺激的下肢收縮時間，用於評估對疼痛的耐受度。根據實驗結果可以證實，使用緩激肽B2受體小干擾核糖核酸會使大鼠對於神經病變痛的耐受性顯著的增加,另外實驗後將大鼠的左右雙側腰椎第四、五及第六背根神經節取出，以RT-PCR檢測緩激肽B2受體的表現量，可以證實在給予小干擾核糖核酸之後緩激肽B2受體的表現量在實驗組有顯著的降低。由以上的結果可以證實緩激肽B2受體小干擾核糖核酸在動物模式可以顯著地降低疼痛的敏感度，達到鎮痛的效果。
第二部分研究的目標是神經母細胞瘤，我們選用人類神經母細胞瘤細胞(IMR-32)做實驗，在加入不同濃度的siRNA後，發現端粒酶的活性顯著降低(加入100 μM濃度時比對照組降低80%活性)，檢測其端粒酶RNA發現加入100 μM組是對照組的20%。
IMR-32細胞的生存能力以WST-1測定法(WST-1 assay)評估，結果發現加入10 μM及100 μM濃度組別的IMR-32細胞生存能力顯著的比對照組低。
細胞凋亡的末期會有細胞核碎裂(fragmentation)，IMR-32細胞培養時加以上不同濃度的端粒酶siRNA，並以DAPI標記，DAPI可以分辨出正常細胞與進行凋亡的細胞，結果發現10 μM及100 μM濃度組的細胞比1 μM濃度及對照組多了10%的凋亡現象。
以流式細胞計量術(flow cytometry)劑量細胞週期不同期的細胞比率，一般而言，癌細胞為不死細胞，因為預估會有較少細胞在sub-G1及G1期；而較多的細胞在S及G2/M期。將IMR-32以不同濃度端粒酶siRNA處置後，結果顯示在10 μM及100 μM濃度組在sub-G1期細胞顯著的增加：在100 μM組則發現G1及S期的細胞顯著的減少。
第三部分研究的目標是視網膜母細胞瘤，我們選用人類視網膜母細胞Y79以及人類視網膜素色上皮細胞(ARPE)進行實驗。在細胞存活能力方面使用MTT-測定法，發現5,10及20 μM濃度的棉酚顯著的抑制Y79的存活能力，但對ARPE細胞沒有影響。
Y79細胞培養時加上不同濃度的棉酚，並以DAPI標記，發現10 μM及20 μM濃度組比對照組有顯著增加的細胞凋亡現象。
以流式細胞計量術計量細胞週期不同期的數目時，發現在10 μM及20 μM時，有顯著的細胞增加在G0/G1期，但在G2/M期則顯著的減少，表示棉酚除了可以誘導凋亡外，也可以使細胞週期停動。
在棉酚導引細胞凋亡機制研究方面，以20 μM濃度組與對照組比較蛋白質表現發現凋亡蛋白質如DR5, p53, Smac, caspase 8, caspase 9 及caspase 3在Y79外細胞組的提升1.5-2倍。而Cytochrome C則提升到5.8倍，顯示棉酚可使用多個機制導引Y79細胞凋亡，包括1) TRAIL-媒介(DR5)途徑，導致caspase家族成員濃度提升，如Smac, Cytochrome C等。2) DNA降解致p53提升及細胞週期停動。
以上的結果，可以推論若能通過未來進一步的臨床試驗，則可以在臨床治療上，使用緩激肽B2受體及端粒酶的小干擾核糖核酸分別治療重度神經疼痛及罹患神經母細胞瘤的病患。另外棉酚除了現有的男性避孕臨床用途外，可以經由臨床試驗將來或能在難治的視網膜母細胞的治療上扮演重要角色。

關鍵字

神經病變痛 ； 緩激肽B2受體 ； 小干擾核糖核酸 ； 端粒酶 ； 神經母細胞瘤 ； 細胞核凝集 ； 細胞週期停動 ； 棉酚 ； 視網膜母細胞瘤 ； 細胞凋亡

並列摘要

RNA interference (RNAi) describes a conserved biological response to double stranded RNA (dsRNA), which results in the degradation of homologous messenger RNA. This process of sequence-specific, post-transcriptional gene silencing has become a key technique for rapidly assessing gene function in both plants and mammals. For target RNA recognition to occur, the small interfering RNA (siRNA) duplex unwinds, allowing binding of one siRNA strand to the target mRNA. The advantage of RNAi to an organism is that siRNA, which specifically binds to target mRNA, prevents damage to other tissues. Adopting this approach may increase a treatment’s therapeutic effect and reduce side effects in patients receiving treatment.
The first part of this dissertation is RNA interference of bradykinin B2 receptor reducing the neuropathic pain caused by sciatic nerve injury. In the condition of cell injury, several inflammatory mediators release from damaged cells. Some of these mediators cause local effect results in increased sensitivity to pain. The hypersensitivity of this sensation is partly due to inflammatory mediators such as prostaglandins, histamine, bradykinin, substance P, and serotonin, which cause a local effect of nociception, and partly some neurotrophic factors. Some mediators transmit the sensation of pain, induced by damage of surrounding cells or even nociceptive neurons. The sensitization of nociception caused by neural damage is normally known as neuropathic pain. Other mediators, such as nerve growth factor and other neurotrophic factors, regulate the progress of neuron regeneration. However, changes in expression of receptors for allogeneic substances such as bradykinin may also be involved, causing a long-term effect. The result shows the nociception caused by neuropathy was reduced by bradykinin B2 receptor siRNA. We therefore supposed that inhibit bradykinin B2 expression may reduce the nociceptive sensation caused by neuropathy.
In our preliminary studies, we screened several inflammatory mediators and found that the nociception caused by neuropathy can be decreased by blocking the transmission of bradykinin. We therefore constructed the RNA interference (RNAi) of bradykinin B2 receptor and applied on the neuropathic animal models. The spared nerve injury models will be used to demonstrate the neuropathic nociception and the mechanical sensitivity behavior test were used to evaluate the degree of neuropathic nociception. Bradykinin B2 receptor expression was upregulated after sciatic nerve crush, while this upregulation was reversed by application of siRNA of bradykinin B2 receptor. This result confirmed that inhibit bradykinin B2 gene expression reduce the nociception caused by neuropathy.
The second part of this dissertation is nuclear condensation and cell cycle arrest induced by telomerase siRNA in neuroblastoma cells. Neuroblastoma is a type of malignant extracranial tumor that occurs in children. Advanced neuroblastoma, and tumors with MYCN amplification in particular, has poor prognoses. Therefore, it is important to find an effective cure for this disease. Small interfering RNA (siRNA) disrupts gene function by specifically binding to target mRNA. In this study, we used siRNA against telomerase to treat neuroblastoma, to evaluate any anti-proliferative effect on these cells. We evaluated cell viability by WST-1 assay on neuroblastoma cells treated with or without telomerase siRNA. Nuclear condensation, an indicator for apoptotic cells, was determined by DAPI labeling following siRNA treatment. The effectiveness of telomerase siRNA on altering the neuroblastoma cell cycle was detected by flow cytometry. Our results indicated that telomerase siRNA reduces the viability of neuroblastoma cells and increases the percentage of cells in the cell cycle’s sub-G1 phase. We found that telomerase siRNA increases the percentage of condensed DNA in neuroblastoma cells. In conclusion, using siRNA against telomerase could be further developed as a therapy for the treatment of neuroblastoma.
The third part of this dissertation is involvement of Smac, p53, and caspase pathways in induction of apoptosis by gossypol in human retinoblastoma cells. Retinoblastoma is a malignant tumor of the retina usually occurring in young children. To date, the conventional treatments for retinoblastoma have been enucleation, cryotherapy, external beam radiotherapy or chemotherapy. Most of these treatments, however, have possible side effects, including blindness, infections, fever, gastrointestinal toxicity and neurotoxicity. More effective treatments are therefore imperative. Gossypol has been reported as a potential inhibitor of cell proliferation in various types of cancers, such as prostate cancer, breast cancer, leukemia, and lung cancer. This study investigates the possible anti-proliferative effect of gossypol on retinoblastoma. The human retinoblastoma cells were cultured with various concentrations of gossypol and checked for cell viability with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nuclear condensation caused by cell apoptosis was detected by staining retinoblastoma cells with 4', 6-diamidino-2-phenylindole (DAPI), counting those with condensed nuclei, and determining the percentage of apoptotic cells. In addition, the stages of apoptosis and phases in cell cycles were examined with flow cytometry. The possible signal transduction pathways involved were examined with a protein array assay and western blot analysis. Our results indicated that after incubation, the cell survival rate was significantly lower after treatment with 5, 10, and 20 μM of gossypol. The maximum antisurvival effect of gossypol was observed at 20 μM, and the number of apoptotic cells was higher in the preparations cultured with 10 and 20 μM of gossypol. The results in flow cytometry indicated that at concentrations of 10 and 20 μM, gossypol increased the proportion of early- and late-apoptotic retinoblastoma cells and induced cell arrest of retinoblastoma cells at the same concentrations. This anti-proliferative effect was later confirmed by upregulating the expression of death receptor 5 (DR5), caspase 8, caspase 9, caspase 3, cytochrome C, tumor protein 53 (p53), and second mitochondria-derived activator of caspases (Smac) in the signal transduction pathways. We concluded that gossypol has an anti-proliferative effect on retinoblastoma cells.

並列關鍵字

Neuropathic pain ； Bradykinin B2 receptor ； siRNA ； Telomerase ； Neuroblastoma ； Nuclear condensation ； Cell cycle arrest ； Gossypol ； Retinoblastoma Apoptosis

參考文獻

1. Abdullah S, Qaddoumi I, Bouffet E. Advances in the management of pediatric central nervous system tumors. Ann N Y Acad Sci. 2008; 1138:22–31

2. Bach LA, Headey SJ, Norton RS. IGF-binding proteins–the pieces are falling into place. Trends Endocrinol Metab. 2005; 16:228-34.

3. Bach LA. IGFBP-6 five years on; not so 'forgotten'? Growth Horm IGF Res. 2005; 15:185-92.

4. Bai SW, Li B, Zhang H, Jonas JB, Zhao B, Shen L, Wang Y. Pax6 Regulates Proliferation and Apoptosis of Human Retinoblastoma Cells. IOVS 2011; 52(7): 4560-70

6. Benz CC, Iyer SB, Asgari HS, Matlin SA, Aronson FR, Barchowsky A. Gossypol effects on endothelial cells and tumor blood flow. Life Sci. 1991; 49:PL67-72.