臺灣博碩士論文加值系統

神經突生長是神經細胞分化與再生的關鍵步驟。一般認為多種化學因素會影響神經細胞的發育，但是近來研究指出物理性因素也會影響，像是外界環境給予的機械力會影響神經幹細胞的分化與神經細胞的發育。然而，到底有哪些基因參與了神經細胞感應外界基質軟硬度仍有許多未知。先前我們研究指出兩個新基因Fam134c與C3orf10分別調控大鼠海馬迴神經元軸突與樹突的生長，Fam134c的產物推測是膜蛋白而C3orf10的產物參與肌動蛋白鏈的分歧。Focal adhesion kinase (FAK) 在非神經細胞中在力感應過程中扮演關鍵的角色。我們假設此兩基因皆參與神經細胞感應外界基質軟硬度，並且是FAK的上/下游訊息分子。本論文利用初級大鼠海馬迴神經元培養於不同軟硬度的聚丙醯胺膠體 (1.0 KPa及7.5 KPa) 與塑膠培養皿上 (Giga Pa)，並利用核醣核酸干擾調降Fam134c與C3orf10的表現，再利用免疫細胞化學法觀察軸突與樹突的生長情形並偵測蛋白質的分佈。首先，發現初級大鼠海馬迴神經元的軸突會隨著軟硬度增加而增加，然而調降Fam134c的表現在軟的基質上可促進軸突的延長，但在硬的基質上則減少軸突的延長；反之，調降C3orf10的表現在硬的基質上會促進初級樹突的延長，但在軟的介質上則不影響。Fam134c與C3orf10在細胞本體的免疫螢光強度都會隨著硬度增加而變少，然而，Fam134c在軸突的免疫螢光強度在軟的基質上則最強；C3orf10在軸突的免疫螢光強度在不同軟硬度下皆相同。另一方面，磷酸化FAK的免疫螢光強度在大鼠海馬迴神經細胞中與纖維母細胞相同，也會隨著硬度增加而增加。Fam134c與C3orf10在細胞的表現位置，在不同軟硬度上都與磷酸化FAK或paxillin的表現位置類似。調降C3orf10的表現在硬的基質抑制了磷酸化FAK的表現量。這些結果顯示Fam134c 和C3orf10分別調控軸突與樹突的生長，並且與FAK作用而參與神經元的力感應。本研究結果將提供重要資訊關於中樞神經元如何感應外界基質軟硬度。

Neurite outgrowth is a critical process of neuronal differentiation. In addition to chemical factors, physical factors, such as mechanical forces imposed by the environment are important to neuronal development. Mechanical properties of extracellular environment have been implicated in differentiation of neuronal stem cells and maturation of neurons. However, which genes are involved in mechanobiology of neurite outgrowth remains largely unknown. Previous studies indicated that two novel genes family with sequence similarity 134 member C (Fam134c) and chromosome 3 open reading frame 10 (C3orf10) differentially regulate axonal and dendritic growth in rat hippocampal neurons. Fam134c is predicted as a membrane protein and C3orf10 is proposed to involve actin nucleation. Focal adhesion kinase (FAK) is key molecule in mechanosensitivity in non-neuronal cells. We hypothesized that Fam134c and C3orf10 involved in mechanbiology of neurite outgrowth under different rigidity of matrix and are up- or down-stream signaling molecules of FAK. RNA interference was used to knockdown Fam134c or C3orf10 of cultured rat hippocampal neurons on different rigidity of polyacrylamide gel and glass dish. Immunocytochemistry was used to observe neuronal morphology and detect protein distribution. Primary rat hippocampal neurons exhibit the rigidity-dependent increases in the length of axons. However, knockdown of Fam134c decreases the length of axons on glass dish, but increases the length of axons on soft gel. In contrast, knockdown of C3orf10 increases the length of primary dendrites on rigid matrix. Immunointensity of both Fam134c and C3orf10 in cell bodies showed rigidity-dependent decrease. However, immunointensity of Fam134c in axons increases on soft gel, but that of C3orf10 has no difference on different rigidity. Immunointensity of phosphorylated FAK (pFAK) exhibits rigidity-dependent increases in primary rat hippocampal neurons. Fam134c and C3orf10 colocalize with pFAK and paxillin on different rigidity of matrix. Knockdown of C3orf10 decreases the expression of pFAK on rigid matrix, but not on soft matrix. These results suggest that Fam134c and C3orf10 differentially modulate axonal and dendritic growth and involve in mechanosensing through the interaction with FAK. Delineation of underlying mechanisms of Fam134c and C3orf10 in mechanobiology of neurons will provide important knowledge how neurons of central nervous system sense the rigidity of extracellular matrix.

Table of contents
中文摘要....... i
Abstract....... iii
誌謝... v
Table of contents...... vii
I. Introduction... 1
1-1. Physiological and pathological importance of rigidity of matrix on brain function. 1
1-2. Physical factors are important to neuronal development.... 1
1-3. Molecules involved in mechanosensing...... 2
1-4. Focal adhesion kinase is a key molecule in mechanosensitivity in non-neuronal cell 3
1-5. Family with sequence similarity 134 member C, FAM134C..... 4
1-6. Chromosome 3 open reading frame 10, C3orf10....... 5
1-7. Hypothesis and specific aims...... 5
II. Materials and methods...... 7
2-1. Cell cultures..... 7
2-2. Plasmid constructs 7
2-3. Preparation of polyacrylamide gels 7
2-4. Recombinant lentivirus production. 8
2-5. Semi-quantitative RT-PCR.. 9
2-6. Primary rat hippocampal neuron culture.... 9
2-7. Immunocytochemistry....... 10
2-8. Measurement of axonal and dendritic parameters.... 10
2-9. Infection. 11
2-10. Measurement of immunointensity in the axon, dendrites and cell body...... 11
2-11. Statistical analysis..... 12
III. Results... 13
3-1. Axonal and dendritic growth of hippocampal neurons on different rigidity of matrix 13
3-2. Novel genes Fam134c and C3orf10 are involved in modulation of mechanosensing in rat hippocampal neurons... 14
3-3. Expression and subcellular localization of Fam134c and C3orf10 of rat hippocampal neurons on different rigidity of matrix.16
3-4. Expression and subcellular localization of pFAK of rat hippocampal neurons on different rigidity of matrix 17
3-5. Fam134c and C3orf10 colocalize with the marker of focal adhesion or pFAK on different rigidity of matrix in primary rat hippocampal neurons....... 17
3-6. C3orf10 is a up-stream signaling molecule of FAK in sensing rigidity of matrix in primary rat hippocampal neurons. 18
IV. Discussion. 20
4-1. Major findings.... 20
4-2. FAK is a mechanosensor in rat hippocampal neurons. 20
4-3. Fam134c involved in modulation of mechanosensing through a dual role in elongation of axons....... 21
4-4. C3orf10 involved in modulation of mechanosensing through negative regulation of primary dendritic elongation on rigid matrix...... 22
V. Conclusion.. 24
VI. References. 25
VII. Figures... 29
Figure 1. Hippocampal neurons exhibit rigidity-dependent increase in the length of axons....... 29
Figure 2. Predicted protein domains of Fam134c. 31
Figure 3. Effects of Fam134c or C3orf10 knockdown on axonal and dendritic growth on different rigidity of matrix at 4 DIV...... 32
Figure 4. Differential effects of Fam134c and C3orf10 knockdown on axonal growth of primary rat hippocampal neurons on different rigidity of matrix.... 34
Figure 5. Differential effects of Fam134c and C3orf10 knockdown on dendritic growth of primary rat hippocampal neurons on different rigidity of matrix. 36
Figure 6. Protein expression and distribution of Fam134c and C3orf10 in cultured rat hippocampal neurons on different rigidity of matrix....... 38
Figure 7. Protein expression level of Fam134c or C3orf10 in cell bodies and axons in primary hippocampal neurons cultured on different rigidity of matrix...... 39
Figure 8. Expression and distribution of pFAK in cultured rat hippocampal neurons on different rigidity of matrix...... 41
Figure 9. Expression of pFAK exhibits rigidity-dependent increases in cell bodies and increases in axons on 7.5 KPa and glass dish..... 42
Figure 10. Fam134c colocalizes with paxillin on different rigidity of matrix in primary rat hippocampal neurons........ 43
Figure 11. C3orf10 colocalizes with paxillin on different rigidity of matrix in primary rat hippocampal neurons........ 44
Figure 12. Fam134c colocalizes with pFAK on different rigidity of matrix in primary rat hippocampal neurons.... 45
Figure 13. C3orf10 colocalizes with pFAK on different rigidity of matrix in primary rat hippocampal neurons.... 46
Figure 14. Effects of C3orf10 knockdown on the level of pFAK in cell bodies of primary rat hippocampal neurons on different rigidity of matrix. 47
Figure 15. Knockdown of C3orf10 increases expression of pFAK on 7.5 KPa gel and glass dish in cell bodies...... 48
Figure 16. Knockdown of C3orf10 increases expression of pFAK on different rigidity of matrix in primary dendrites...... 49
Figure 17. Proposed mechanisms of Fam134c and C3orf10 in modulation of mechanosensing in hippocampal neruons... 51
VIII. Appendix. 52
Table 1. The clone list of shRNAs...... 52
Table 2. Primer sequences and amplicon sizes for semi-quantitative RT-PCR experiments.. 53

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