CONCLUSION - In vitro cytotoxicity of N. fowleri transfected with a RNAi vector

F. In vitro cytotoxicity of N. fowleri transfected with a RNAi vector

V. CONCLUSION

In the present study, to observe whether the nfa1 gene could be related with in vitro cytotoxicity, an nfa1 gene and Nfa1 protein were knockdowned in pathogenic N.

fowleri using RNAi strategy. By synthetic dsRNA of the nfa1 gene ORF, the expression of nfa1 gene and the Nfa1 protein were knockdowned about 50% and 30%, respectively. However, the exoression of nfa1 gene and the Nfa1 protein by in vitro transcribed asRNA were not highly knockdowned as dsRNA of nfa1 gene. Four synthetic siRNAs were not act equally, but sinfa1-1 was the highest effective for knockdown of the expression of nfa1 gene and Nfa1 protein with 70% and 43%, respectively. However, N. fowleri trophozoites transfected with synthetic dsRNA or sinfa1-1 did not highly induce in vitro cytotoxicity against murine macrophages as compared with normal N. fowleri trophozoites. Therefore, a vector-based system, in which transfected genes can be maintain longer, was used to transfect the nfa1 gene into N. fowleri. A pAct/SAGAH vector with sinfa1-1 and a pAct/asnfa1AGAH vector with asRNA of the nfa1 gene ORF to transcribe efficiently selective marker genes were cloned and transfected into N. fowleri. The expression of nfa1 gene and Nfa1 protein were efficiently knockdowned about 60% and 29%, respectively by a pAct/SAGAH vector, as compared with a pAct/asnfa1AGAH vector of 30% in the nfa1 gene and 18% in Nfa1 protein. In particular, the in vitro cytotoxicity of N.

fowleri transfected with a pAct/SAGAH vector against macrophages were decreased

with 26.6% at 17 h and 26.8% at 24 h in comparision with normal N. fowleri and N.

fowleri transfected with a pAct/AGAH control vector. Also, the in vitro cytotoxicity by a pAct/asnfa1AGAH vector was decreased with 7.4% at 17 h and 6.6% at 24 h.

These results suggest that the mechanism of RNAi should be worked in N. fowleri trophozoites. Therefore, single stranded RNA, dsRNA, siRNA and siRNA-vector were not only efficiently transfected into N. fowleri using each transfection reagent but also the Nfa1 protein play very important role in destroying macrophages.

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–국문요약–

Antisense RNA를를 이용한를를 이용한이용한이용한 병원성병원성병원성자유아메바로부터병원성자유아메바로부터자유아메바로부터자유아메바로부터 클로닝된클로닝된클로닝된클로닝된 nfa1 유전자의유전자의유전자의 발현억제유전자의 발현억제발현억제 시스템발현억제 시스템시스템 시스템

아주대학교 대학원의학과 이 상 철

(지도교수: 신 호 준)

파울러자유아메바 (Naegleria fowleri)에 의한 원발성 아메바성 수막뇌염 (primary amoebic meningoencephalitis)은 소아나 실험동물에서 급성으로 발병하며 치명적 이다. 파울러자유아메바의 병인기전 및 독성과 연관된 단백질에 관한 보고는 거의 미약하다. 또한, 단백질의 기능을 파악하기 위한 transfection 시스템도 아직 완전히 확립되지 못하였다. 본 연구에서 사용된 병원성 nfa1 유전자는 병원성 파울러자유아메바로부터 immunoscreening 통해 클로닝되었으며, 357 개의 DNA로 구성되어 있다. Nfa1 단백질은 아메바의 위족 특히, food-cup 형성에 관여하는 것으로 알려져 있다. 또한 anti-Nfa1 항체는 파울러아메바의 세포독성을 감소 시키는 효과를 갖고 있다. 따라서 본 연구자는 nfa1 유전자에 대한 antisense RNA와 siRNA를 이용하여, transfection system의 확립과 더불어 표적세포에 대한 아메바의 세포독성에 있어서 Nfa1 단백질의 연관성을 확인하고자 하였다. 시험관 내에서 합성된 dsRNA에 의해서 nfa1 유전자의발현과 Nfa1 단백질의 생산은 각각 약 50% 및 30%가 억제되었다. 하지만, 시험관 내에서 합성된 antisense RNA에 의해서는 dsRNA에 의한 효과만큼 크지는 않았다. 합성된 네개의 siRNAs들 중 sinfa1-1이 최대의 효과가 있었으며, nfa1 유전자에는 70% 그리고 Nfa1 단백질에

는 43%의 억제를 보여주었다. 하지만, 합성된 dsRNA 혹은 sinfa1-1을 transfection 한 파울러자유아메바는 대식세포에 대한 세포독성을 유발하지 않았다. 따라서, transfection 된 유전자가 더욱 더 세포내에서 오랫동안 유지 될 수 있는 벡터를 이용하여 파울러자유아메바에 transfection하고 세포독성을 관찰하고자 하였다.

sinfa1-1이 클로닝된 pAct/SAGAH와 antisense nfa1 유전자 mRNA가 클로닝된 pAct/asnfa1AGAH 벡터가 이용되었다. pAct/SAGAH 벡터에 의해서 nfa1 유전자의 발현과 Nfa1 단백질 생산이 각각 60% 및 29%가 억제되었고, pAct/asnfa1AGAH 벡터에 의해서 유전자와 단백질이 30% 및 18%가 각각 억제되었다. 특히, pAct/SAGAH 벡터가 transfection 된 파울러자유아메바는 표적세포인 대식세포와 17 시간 혼합배양에 있어서 대조군들보다 약 26.6% 그리고 24 시간에 있어서 약 26.8%의 세포독성 억제를 보여주었다. pAct/asnfa1AGAH 벡터가 transfection 된

문서에서 Knockdown of nfa1 Gene Cloned from Naegleria fowleri by Antisense RNA (페이지 70-85)