ヒト血清点眼におけるリゾチーム遺伝子発現の定量

Quantification of lysozyme gene expression in eye drops of human serum

要約　目的：細菌の細胞壁多糖類を加水分解して，溶菌作用を有する酵素であるリゾチーム遺伝子（Lyz）の発現量についてリアルタイムpolymerase chain reaction（PCR）によりヒト血清点眼において定量を試みた結果の報告。

対象と方法：定量のためLyzのcDNAは増殖された大腸菌のプラスミドから10×10⁷のコピーを得た。希釈倍率は1，4，16，256，1,024倍とした。ヒト血清は原液，4倍希釈，レボフロキサシン点眼液（Le）を添加したものからRNAを抽出し，逆転写を行った。微量分光光度計により，RNA，DNA量を測り，cDNA，プライマー，MyGo-greenを混合し，Lyz発現をMyGoリアルタイムPCRにより定量的に解析した。

結果：LyzのcDNAコピー数（q）に対する検量線はCq（増殖曲線がthreshold lineと交差するときのcycle数）＝−2.62 log10（q）＋37.27，r²＝0.999であった。また，Tm値（二本鎖DNAの50％が一本鎖に解離するときの温度）はコントロールとヒト血清との間で有意差はなく，ヒト血清の原液は420.4±172.6コピー/mLという量であった。RNAのみ原液は4倍希釈より有意に多く，DNA，Lyz発現量では原液，4倍希釈，Leを添加したものの間で有意差を認めなかった。

結論：Lyz発現量はヒト血清点眼と原液との間で有意差はなく，定量化の応用範囲を広げられる可能性があると思われた。

Abstract　Purpose：To quantify the expression of human lysozyme gene（Lyz）in human serum as medicated eye drops for dry eye using a real time reverse-transcriptase polymerase chain reaction（RT-PCR）.

Materials and Methods：We obtained 10×10⁷ copies of Lyz cDNA in the plasmid by the proliferation of Escherichia coli bacteria. Dilution factors as linear regression for quantification were set up as 4, 16, 256, and 1,024 times. We extracted total RNA in human serum, and cDNA was synthesized. We designed the primers for RT-PCR amplification of the Lyz cDNA. The reactions were carried out at the following temperatures：95℃ for denaturation and 60℃ for annealing）. After mixing the cDNA, primer, and MyGo-green, the expression of Lyz cDNA was measured using the MyGo real time of PCR system. The technology of this system is extremely innovative and enables a rapid and simultaneous evaluation PCR experiments. Fluorometric analysis of the formed PCR products was performed as a real-time measurement either continuously or specifically defined time points during each PCR cycle.

Results：The equation of quantification for the Lyz cDNA expression is shown（q：copies of Lyz cDNA, Cq：number of cycles when logarithm amplification started, equation：Cq＝−2.62 log₁₀（q）＋37.27, r²＝0.999）. There were no significant differences in Tm value（temperature during melting curve when 50％ of double chains were changed into single chains）between human Lyz cDNA and cDNA from human serum（420.4±172.6 copies/mL）. Total RNA of human serum without dilution was significantly decreased by 4 times dilution. There were no significant differences in total DNA and Lyz cDNA expression among serum without dilution, that diluted at 4 times, and serum with antibiotics.

Conclusions：It is possible for Lyz cDNA expression of human serum to be quantified by real time PCR system, and these studies concerned with Lyz cDNA expression could be applied about eye drops using human serum.