ΠŸΠΎΠΌΠΎΡ‰ΡŒ Π² ΡƒΡ‡Ρ‘Π±Π΅, ΠΎΡ‡Π΅Π½ΡŒ быстро...
Π Π°Π±ΠΎΡ‚Π°Π΅ΠΌ вмСстС Π΄ΠΎ ΠΏΠΎΠ±Π΅Π΄Ρ‹

ΠŸΠΎΠ»ΡƒΡ‡Π΅Π½ΠΈΠ΅ гСнСтичСски ΠΊΠΎΠ΄ΠΈΡ€ΡƒΠ΅ΠΌΡ‹Ρ… FRET-сСнсоров каспазы-3 Π½Π° основС Ρ‚Π΅Ρ€Π±ΠΈΠΉ-ΡΠ²ΡΠ·Ρ‹Π²Π°ΡŽΡ‰Π΅Π³ΠΎ ΠΏΠ΅ΠΏΡ‚ΠΈΠ΄Π° ΠΈ красных флуорСсцСнтных Π±Π΅Π»ΠΊΠΎΠ² DsRed2 ΠΈ TagRFP

Π”ΠΈΡΡΠ΅Ρ€Ρ‚Π°Ρ†ΠΈΡΠŸΠΎΠΌΠΎΡ‰ΡŒ Π² Π½Π°ΠΏΠΈΡΠ°Π½ΠΈΠΈΠ£Π·Π½Π°Ρ‚ΡŒ ΡΡ‚ΠΎΠΈΠΌΠΎΡΡ‚ΡŒΠΌΠΎΠ΅ΠΉ Ρ€Π°Π±ΠΎΡ‚Ρ‹

Π’Π΅ΠΌ Π½Π΅ ΠΌΠ΅Π½Π΅Π΅, ΠΏΡ€ΠΎΠ±Π»Π΅ΠΌΠ° Ρ„ΠΎΠ½ΠΎΠ²ΠΎΠ³ΠΎ сигнала ΠΊΠ»Π΅Ρ‚ΠΎΠΊ, связанная с Π°Π²Ρ‚офлуорСсцСнциСй Π±ΠΈΠΎΠΌΠΎΠ»Π΅ΠΊΡƒΠ» ΠΈ ΡΠ²Π΅Ρ‚орассСяниСм остаСтся ΠΎΡ‚ΠΊΡ€Ρ‹Ρ‚ΠΎΠΉ. Для Ρ€Π΅ΡˆΠ΅Π½ΠΈΡ этой ΠΏΡ€ΠΎΠ±Π»Π΅ΠΌΡ‹ Ρ€Π°Π·Ρ€Π°Π±Π°Ρ‚Ρ‹Π²Π°ΡŽΡ‚ сСнсоры Π½Π° ΠΎΡΠ½ΠΎΠ²Π΅ Π±Π΅Π»ΠΊΠΎΠ² со ΡΠΏΠ΅ΠΊΡ‚Ρ€ΠΎΠΌ флуорСсцСнции Π² ΠΊΡ€Π°ΡΠ½ΠΎΠΉ ΠΈ Π΄Π°Π»ΡŒΠ½Π΅ΠΊΡ€Π°ΡΠ½ΠΎΠΉ области. Π”Ρ€ΡƒΠ³ΠΈΠΌ ΠΏΠΎΠ΄Ρ…ΠΎΠ΄ΠΎΠΌ являСтся использованиС Π² ΠΊΠ°Ρ‡Π΅ΡΡ‚Π²Π΅ Π΄ΠΎΠ½ΠΎΡ€ΠΎΠ² Π²ΠΎ Π‘Π˜Π•Π’-ΠΏΠ°Ρ€Π΅ Ρ„Π»ΡƒΠΎΡ€Π΅ΡΡ†ΠΈΡ€ΡƒΡŽΡ‰ΠΈΡ… комплСксов Π»Π°Π½Ρ‚Π°Π½ΠΈΠ΄ΠΎΠ² с ΠΌΠΈΠΊΡ€ΠΎΠΈ миллисСкундным Π²Ρ€Π΅ΠΌΠ΅Π½Π΅ΠΌ… Π§ΠΈΡ‚Π°Ρ‚ΡŒ Π΅Ρ‰Ρ‘ >

Π‘ΠΎΠ΄Π΅Ρ€ΠΆΠ°Π½ΠΈΠ΅

  • БПИБОК Π‘ΠžΠšΠ ΠΠ©Π•ΠΠ˜Π™
  • ГЛАВА 1. ΠžΠ‘Π—ΠžΠ  Π›Π˜Π’Π•Π ΠΠ’Π£Π Π«
    • 1. 1. Π˜Π·ΡƒΡ‡Π΅Π½ΠΈΠ΅ Ρ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚Π°Ρ‚ΠΈΠ²Π½ΠΎΠΉ активности каспазы-3 с ΠΏΠΎΠΌΠΎΡ‰ΡŒΡŽ FRET-биосСнсоров
    • 1. 2. ΠŸΡ€ΠΎΠ±Π»Π΅ΠΌΠ° Ρ„ΠΎΠ½ΠΎΠ²ΠΎΠΉ флуорСсцСнции
    • 1. 3. ЀлуорСсцСнтныС свойства Π»Π°Π½Ρ‚Π°Π½ΠΈΠ΄ΠΎΠ². БСнсибилизация флуорСсцСнции. Π›Π°Π½Ρ‚Π°Π½ΠΈΠ΄-ΡΠ²ΡΠ·Ρ‹Π²Π°ΡŽΡ‰ΠΈΠ΅ ΠΏΠ΅ΠΏΡ‚ΠΈΠ΄Ρ‹
    • 1. 4. ΠœΠ½ΠΎΠ³ΠΎΡ„ΠΎΡ‚ΠΎΠ½Π½ΠΎΠ΅ Π²ΠΎΠ·Π±ΡƒΠΆΠ΄Π΅Π½ΠΈΠ΅ Π»Π°Π½Ρ‚Π°Π½ΠΈΠ΄ΠΎΠ²
    • 1. 5. ΠžΡΠ½ΠΎΠ²Π½Ρ‹Π΅ Ρ‚Π΅Π½Π΄Π΅Π½Ρ†ΠΈΠΈ изучСния ΠΈΠ½Π΄ΡƒΠΊΡ‚ΠΈΠ²Π½ΠΎ-рСзонансного пСрСноса энСргии
    • 1. 6. ΠšΠΎΠΌΠΏΠ»Π΅ΠΊΡΠ½Ρ‹Π΅ соСдинСния Π»Π°Π½Ρ‚Π°Π½ΠΈΠ΄ΠΎΠ² Π² ΠΊΠ°Ρ‡Π΅ΡΡ‚Π²Π΅ Π΄ΠΎΠ½ΠΎΡ€ΠΎΠ² Π²ΠΎ FRET-ΠΏΠ°Ρ€Π°Ρ… ΠΏΡ€ΠΈ ΠΈΠ·ΡƒΡ‡Π΅Π½ΠΈΠΈ биологичСских процСссов ΠΈ ΠΏΡ€ΠΎΠ²Π΅Π΄Π΅Π½ΠΈΠΈ биоаналитичСских Ρ€Π΅Π°ΠΊΡ†ΠΈΠΉ
  • ГЛАВА 2. ΠœΠΠ’Π•Π Π˜ΠΠ›Π« И ΠœΠ•Π’ΠžΠ”Π«
    • 2. 1. ΠžΠ±ΠΎΡ€ΡƒΠ΄ΠΎΠ²Π°Π½ΠΈΠ΅
    • 2. 2. Π Π΅Π°ΠΊΡ‚ΠΈΠ²Ρ‹
    • 2. 3. ΠœΠ΅Ρ‚ΠΎΠ΄Ρ‹ исслСдования
  • ГЛАВА 3. РЕЗУЛЬВАВЫ И ΠžΠ‘Π‘Π£Π–Π”Π•ΠΠ˜Π•
    • 3. 1. Π₯арактСристика экспрСссии флуорСсцСнтных Π±Π΅Π»ΠΊΠΎΠ² Π² ΡƒΡΠ»ΠΎΠ²ΠΈΡΡ… in vitro ΠΈ in vivo
    • 3. 2. ΠŸΠΎΠ»ΡƒΡ‡Π΅Π½ΠΈΠ΅ гСнСтичСски ΠΊΠΎΠ΄ΠΈΡ€ΡƒΠ΅ΠΌΡ‹Ρ… конструкций, ΠΊΠΎΠ΄ΠΈΡ€ΡƒΡŽΡ‰ΠΈΡ… Π³ΠΈΠ±Ρ€ΠΈΠ΄Π½Ρ‹Π΅ Π±Π΅Π»ΠΊΠΈ. Π³
    • 3. 3. ΠŸΠΎΠ»ΡƒΡ‡Π΅Π½ΠΈΠ΅ высокоочищСнных Ρ„Ρ€Π°ΠΊΡ†ΠΈΠΉ Π³ΠΈΠ±Ρ€ΠΈΠ΄Π½Ρ‹Ρ… Π±Π΅Π»ΠΊΠΎΠ² ΠΈ ΠΎΠΏΡ€Π΅Π΄Π΅Π»Π΅Π½ΠΈΠ΅ ΠΈΡ… Π°Π³Ρ€Π΅Π³Π°Ρ‚Π½ΠΎΠ³ΠΎ состояния
    • 3. 4. Π˜Π·ΡƒΡ‡Π΅Π½ΠΈΠ΅ пСрСноса энСргии
    • 3. 5. ΠžΠΏΡ€Π΅Π΄Π΅Π»Π΅Π½ΠΈΠ΅ активности каспазы-Π—

ΠŸΠΎΠ»ΡƒΡ‡Π΅Π½ΠΈΠ΅ гСнСтичСски ΠΊΠΎΠ΄ΠΈΡ€ΡƒΠ΅ΠΌΡ‹Ρ… FRET-сСнсоров каспазы-3 Π½Π° основС Ρ‚Π΅Ρ€Π±ΠΈΠΉ-ΡΠ²ΡΠ·Ρ‹Π²Π°ΡŽΡ‰Π΅Π³ΠΎ ΠΏΠ΅ΠΏΡ‚ΠΈΠ΄Π° ΠΈ красных флуорСсцСнтных Π±Π΅Π»ΠΊΠΎΠ² DsRed2 ΠΈ TagRFP (Ρ€Π΅Ρ„Π΅Ρ€Π°Ρ‚, курсовая, Π΄ΠΈΠΏΠ»ΠΎΠΌ, ΠΊΠΎΠ½Ρ‚Ρ€ΠΎΠ»ΡŒΠ½Π°Ρ)

Апоптоз являСтся ΠΎΠ΄Π½ΠΈΠΌ ΠΈΠ· Π²Π°Ρ€ΠΈΠ°Π½Ρ‚ΠΎΠ² ΠΏΡ€ΠΎΠ³Ρ€Π°ΠΌΠΌΠΈΡ€ΠΎΠ²Π°Π½Π½ΠΎΠΉ Π³ΠΈΠ±Π΅Π»ΠΈ ΠΊΠ»Π΅Ρ‚ΠΎΠΊ. Он ΡƒΡ‡Π°ΡΡ‚Π²ΡƒΠ΅Ρ‚ Π² Π³ΠΈΡΡ‚ΠΎΠ³Π΅Π½Π΅Π·Π΅ ΠΈ ΠΏΠΎΠ΄Π΄Π΅Ρ€ΠΆΠ°Π½ΠΈΠΈ гомСостаза ΠΎΡ€Π³Π°Π½ΠΈΠ·ΠΌΠ°. ΠΠ°Ρ€ΡƒΡˆΠ΅Π½ΠΈΡ Π°ΠΏΠΎΠΏΡ‚ΠΎΠ·Π° приводят ΠΊ Ρ€Π°Π·Π²ΠΈΡ‚ΠΈΡŽ Π½Π΅ΠΉΡ€ΠΎΠ΄Π΅Π³Π΅Π½Π΅Ρ€Π°Ρ‚ΠΈΠ²Π½Ρ‹Ρ…, ΠΎΠΏΡƒΡ…ΠΎΠ»Π΅Π²Ρ‹Ρ…, Π°ΡƒΡ‚ΠΎΠΈΠΌΠΌΡƒΠ½Π½Ρ‹Ρ… Π·Π°Π±ΠΎΠ»Π΅Π²Π°Π½ΠΈΠΉ. Π—Π° Π·Π°ΠΏΡƒΡΠΊ протСолитичСского каскада Π°ΠΏΠΎΠΏΡ‚ΠΎΠ·Π° ΠΎΡ‚Π²Π΅Ρ‡Π°ΡŽΡ‚ ΠΈΠ½ΠΈΡ†ΠΈΠ°Ρ‚ΠΎΡ€Π½Ρ‹Π΅ ΠΈ ΡΡ„Ρ„Π΅ΠΊΡ‚ΠΎΡ€Π½Ρ‹Π΅ каспазы. Π‘Ρ€Π΅Π΄ΠΈ Π½ΠΈΡ… каспаза-3 являСтся Ρ†Π΅Π½Ρ‚Ρ€Π°Π»ΡŒΠ½Ρ‹ΠΌ Ρ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚ΠΎΠΌ Π°ΠΏΠΎΠΏΡ‚ΠΎΠ·Π°, Ρ‚Π°ΠΊ ΠΊΠ°ΠΊ Π½Π° Π½Π΅ΠΉ сходятся Ρ€Π΅Ρ†Π΅ΠΏΡ‚ΠΎΡ€Π½Ρ‹ΠΉ ΠΈ ΠΌΠΈΡ‚ΠΎΡ…ΠΎΠ½Π΄Ρ€ΠΈΠ°Π»ΡŒΠ½Ρ‹ΠΉ ΠΏΡƒΡ‚ΠΈ Π°ΠΊΡ‚ΠΈΠ²Π°Ρ†ΠΈΠΈ протСолитичСского каскада [1]. Π’ Π°ΠΊΡ‚ΠΈΠ²Π½ΠΎΠΌ состоянии ΠΎΠ½Π° ΠΈΠ½ΠΈΡ†ΠΈΠΈΡ€ΡƒΠ΅Ρ‚ Π°ΠΊΡ‚ΠΈΠ²Π°Ρ†ΠΈΡŽ Π΄Ρ€ΡƒΠ³ΠΈΡ… эффСкторных каспаз ΠΈ Π³ΠΈΠ΄Ρ€ΠΎΠ»ΠΈΠ·ΡƒΠ΅Ρ‚ Ρ€Π°Π·Π»ΠΈΡ‡Π½Ρ‹Π΅ ΠΊΠ»Π΅Ρ‚ΠΎΡ‡Π½Ρ‹Π΅ субстраты [2]. Π€Π΅Ρ€ΠΌΠ΅Π½Ρ‚ прСдставляСт интСрСс ΠΊΠ°ΠΊ тСрапСвтичСская мишСнь ΠΏΡ€ΠΈ воздСйствии Π½Π° ΠΊΠ»Π΅Ρ‚ΠΊΡƒ Ρ€Π°Π·Π»ΠΈΡ‡Π½Ρ‹Ρ… лСкарствСнных ΠΏΡ€Π΅ΠΏΠ°Ρ€Π°Ρ‚ΠΎΠ², ΠΈΠ½Π΄ΡƒΡ†ΠΈΡ€ΡƒΡŽΡ‰ΠΈΡ… Π°ΠΏΠΎΠΏΡ‚ΠΎΠ·. Π£Ρ€ΠΎΠ²Π΅Π½ΡŒ активности каспазы-3 являСтся Π²Π°ΠΆΠ½Ρ‹ΠΌ прогностичСским ΠΌΠ°Ρ€ΠΊΠ΅Ρ€ΠΎΠΌ для ΠΎΡ†Π΅Π½ΠΊΠΈ агрСссивности патологичСских процСссов ΠΈ ΡΡ„фСктивности дСйствия лСкарствСнных срСдств.

Для изучСния Ρ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚Π°Ρ‚ΠΈΠ²Π½ΠΎΠΉ активности Π² ΠΆΠΈΠ²Ρ‹Ρ… ΠΊΠ»Π΅Ρ‚ΠΊΠ°Ρ… Π² ΠΏΠΎΡΠ»Π΅Π΄Π½Π΅Π΅ врСмя Π°ΠΊΡ‚ΠΈΠ²Π½ΠΎ Ρ€Π°Π·Ρ€Π°Π±Π°Ρ‚Ρ‹Π²Π°ΡŽΡ‚ΡΡ FRET-биосСнсоры, Π² ΠΎΡΠ½ΠΎΠ²Π΅ дСйствия ΠΊΠΎΡ‚ΠΎΡ€Ρ‹Ρ… Π»Π΅ΠΆΠΈΡ‚ ΠΌΠ΅Ρ‚ΠΎΠ΄ ΠΈΠ½Π΄ΡƒΠΊΡ‚ΠΈΠ²Π½ΠΎ-рСзонансного пСрСноса энСргии [3]. Π’ Π΄Π°Π½Π½Ρ‹Ρ… сСнсорах FRET проявляСтся ΠΊΠ°ΠΊ динамичСский Ρ‚ΠΈΠΏ Ρ‚ΡƒΡˆΠ΅Π½ΠΈΡ флуорСсцСнции Π΄ΠΎΠ½ΠΎΡ€Π° ΠΈ, соотвСтствСнно, характСризуСтся сниТСниСм Π²Ρ€Π΅ΠΌΠ΅Π½ΠΈ ΠΆΠΈΠ·Π½ΠΈ Π΄ΠΎΠ½ΠΎΡ€Π° Π² Π²ΠΎΠ·Π±ΡƒΠΆΠ΄Π΅Π½Π½ΠΎΠΌ состоянии [4]. По ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΡŽ ΠΏΠ°Ρ€Π°ΠΌΠ΅Ρ‚Ρ€ΠΎΠ² FRET ΠΌΠΎΠΆΠ½ΠΎ ΠΏΡ€ΠΎΠ²ΠΎΠ΄ΠΈΡ‚ΡŒ прямой ΠΌΠΎΠ½ΠΈΡ‚ΠΎΡ€ΠΈΠ½Π³ Ρ„Π΅Ρ€ΠΌΠ΅Π½Ρ‚Π°Ρ‚ΠΈΠ²Π½ΠΎΠΉ активности, Π±Π΅Π»ΠΎΠΊ-Π±Π΅Π»ΠΊΠΎΠ²Ρ‹Ρ… взаимодСйствий, измСнСния ΠΊΠΎΠ½Ρ„ΠΎΡ€ΠΌΠ°Ρ†ΠΈΠΈ Π±Π΅Π»ΠΊΠΎΠ² ΠΈ Π΄Ρ€. [5].

Π’ Π½Π°ΡΡ‚оящСС врСмя Π°ΠΊΡ‚ΠΈΠ²Π½ΠΎ вСдСтся Ρ€Π°Π·Ρ€Π°Π±ΠΎΡ‚ΠΊΠ° FRET-биосСнсоров, Π³Π΄Π΅ Π² ΠΊΠ°Ρ‡Π΅ΡΡ‚Π²Π΅ Π΄ΠΎΠ½ΠΎΡ€ΠΎΠ² ΠΈ Π°ΠΊΡ†Π΅ΠΏΡ‚ΠΎΡ€ΠΎΠ² ΠΈΡΠΏΠΎΠ»ΡŒΠ·ΡƒΡŽΡ‚ΡΡ флуорСсцСнтныС Π±Π΅Π»ΠΊΠΈ [Π±]. ЭкспрСссия Π½ΡƒΠΊΠ»Π΅ΠΎΡ‚ΠΈΠ΄Π½ΠΎΠΉ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°Ρ‚Π΅Π»ΡŒΠ½ΠΎΡΡ‚ΠΈ флуорСсцСнтных Π±Π΅Π»ΠΊΠΎΠ² ΠΈ ΡΡƒΠ±ΡΡ‚Ρ€Π°Ρ‚Π° каспазы-3 Π² Π΅Π΄ΠΈΠ½ΠΎΠΉ Ρ€Π°ΠΌΠΊΠ΅ считывания являСтся основой Ρ€Π°Π·Ρ€Π°Π±ΠΎΡ‚ΠΊΠΈ гСнСтичСски ΠΊΠΎΠ΄ΠΈΡ€ΡƒΠ΅ΠΌΡ‹Ρ… сСнсоров каспазной активности. Π˜Π½Π΄ΡƒΠΊΡ‚ΠΈΠ²Π½ΠΎ-рСзонансный пСрСнос энСргии Π² Π΄Π°Π½Π½Ρ‹Ρ… сСнсорах проявляСтся ΠΊΠ°ΠΊ динамичСский Ρ‚ΠΈΠΏ Ρ‚ΡƒΡˆΠ΅Π½ΠΈΡ флуорСсцСнции Π΄ΠΎΠ½ΠΎΡ€Π° ΠΈ, соотвСтствСнно, характСризуСтся сниТСниСм Π²Ρ€Π΅ΠΌΠ΅Π½ΠΈ ΠΆΠΈΠ·Π½ΠΈ Π΄ΠΎΠ½ΠΎΡ€Π° Π² Π²ΠΎΠ·Π±ΡƒΠΆΠ΄Π΅Π½Π½ΠΎΠΌ состоянии. Π“ΠΈΠ΄Ρ€ΠΎΠ»ΠΈΠ· Π»ΠΈΠ½ΠΊΠ΅Ρ€Π°, содСрТащСго Π°ΠΌΠΈΠ½ΠΎΠΊΠΈΡΠ»ΠΎΡ‚Π½ΡƒΡŽ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°Ρ‚Π΅Π»ΡŒΠ½ΠΎΡΡ‚ΡŒ DEXD, спСцифичСски распознаваСмой каспазой-3, ΠΏΡ€ΠΈΠ²ΠΎΠ΄ΠΈΡ‚ ΠΊ Ρ„изичСскому Ρ€Π°Π·Π΄Π΅Π»Π΅Π½ΠΈΡŽ Π΄ΠΎΠ½ΠΎΡ€Π° ΠΈ Π°ΠΊΡ†Π΅ΠΏΡ‚ΠΎΡ€Π°, Π² Ρ€Π΅Π·ΡƒΠ»ΡŒΡ‚Π°Ρ‚Π΅ Ρ‡Π΅Π³ΠΎ условия пСрСноса энСргии Π½Π°Ρ€ΡƒΡˆΠ°ΡŽΡ‚ΡΡ ΠΈ, соотвСтствСнно, снимаСтся Ρ‚ΡƒΡˆΠ΅Π½ΠΈΠ΅ [7]. Π”Π°Π½Π½Ρ‹Π΅ сСнсоры ΠΎΠ±Π»Π°Π΄Π°ΡŽΡ‚ прСимущСствами ΠΏΠΎ ΡΡ€Π°Π²Π½Π΅Π½ΠΈΡŽ с ΡΠΈΠ½Ρ‚СтичСскими Ρ„Π»ΡƒΠΎΡ€ΠΎΠ³Π΅Π½Π½Ρ‹ΠΌΠΈ субстратами. Π­Ρ‚ΠΎ связано с Ρ‚Π΅ΠΌ, Ρ‡Ρ‚ΠΎ гСнСтичСски ΠΊΠΎΠ΄ΠΈΡ€ΡƒΠ΅ΠΌΡ‹Π΅ сСнсоры ΡΠΊΡΠΏΡ€Π΅ΡΡΠΈΡ€ΡƒΡŽΡ‚ΡΡ ' самой ΠΊΠ»Π΅Ρ‚ΠΊΠΎΠΉ ΠΈ Π½Π΅ Ρ‚Ρ€Π΅Π±ΡƒΡŽΡ‚ внСшнСй ΠΈΠ½ΡŠΠ΅ΠΊΡ†ΠΈΠΈ, ΠΊΡ€ΠΎΠΌΠ΅ Ρ‚ΠΎΠ³ΠΎ ΠΎΠ½ΠΈ ΡΠ²Π»ΡΡŽΡ‚ΡΡ высокостандартизованными ΠΌΠ΅Ρ‚ΠΊΠ°ΠΌΠΈ со ΡΡ‚Ρ€ΠΎΠ³ΠΎ ΠΎΠΏΡ€Π΅Π΄Π΅Π»Π΅Π½Π½Ρ‹ΠΌΠΈ Ρ‚ΠΎΡ‡ΠΊΠ°ΠΌΠΈ присоСдинСния.

Π’Π΅ΠΌ Π½Π΅ ΠΌΠ΅Π½Π΅Π΅, ΠΏΡ€ΠΎΠ±Π»Π΅ΠΌΠ° Ρ„ΠΎΠ½ΠΎΠ²ΠΎΠ³ΠΎ сигнала ΠΊΠ»Π΅Ρ‚ΠΎΠΊ, связанная с Π°Π²Ρ‚офлуорСсцСнциСй Π±ΠΈΠΎΠΌΠΎΠ»Π΅ΠΊΡƒΠ» ΠΈ ΡΠ²Π΅Ρ‚орассСяниСм остаСтся ΠΎΡ‚ΠΊΡ€Ρ‹Ρ‚ΠΎΠΉ. Для Ρ€Π΅ΡˆΠ΅Π½ΠΈΡ этой ΠΏΡ€ΠΎΠ±Π»Π΅ΠΌΡ‹ Ρ€Π°Π·Ρ€Π°Π±Π°Ρ‚Ρ‹Π²Π°ΡŽΡ‚ сСнсоры Π½Π° ΠΎΡΠ½ΠΎΠ²Π΅ Π±Π΅Π»ΠΊΠΎΠ² со ΡΠΏΠ΅ΠΊΡ‚Ρ€ΠΎΠΌ флуорСсцСнции Π² ΠΊΡ€Π°ΡΠ½ΠΎΠΉ ΠΈ Π΄Π°Π»ΡŒΠ½Π΅ΠΊΡ€Π°ΡΠ½ΠΎΠΉ области [8, 9, 10]. Π”Ρ€ΡƒΠ³ΠΈΠΌ ΠΏΠΎΠ΄Ρ…ΠΎΠ΄ΠΎΠΌ являСтся использованиС Π² ΠΊΠ°Ρ‡Π΅ΡΡ‚Π²Π΅ Π΄ΠΎΠ½ΠΎΡ€ΠΎΠ² Π²ΠΎ Π‘Π˜Π•Π’-ΠΏΠ°Ρ€Π΅ Ρ„Π»ΡƒΠΎΡ€Π΅ΡΡ†ΠΈΡ€ΡƒΡŽΡ‰ΠΈΡ… комплСксов Π»Π°Π½Ρ‚Π°Π½ΠΈΠ΄ΠΎΠ² с ΠΌΠΈΠΊΡ€ΠΎΠΈ миллисСкундным Π²Ρ€Π΅ΠΌΠ΅Π½Π΅ΠΌ ΠΆΠΈΠ·Π½ΠΈ Π² Π²ΠΎΠ·Π±ΡƒΠΆΠ΄Π΅Π½Π½ΠΎΠΌ состоянии [11]. ΠŸΡ€ΠΈ пСрСносС энСргии ΠΎΡ‚ Π΄ΠΎΠ½ΠΎΡ€Π°-Π»Π°Π½Ρ‚Π°Π½ΠΈΠ΄Π° врСмя ΠΆΠΈΠ·Π½ΠΈ Π²ΠΎΠ·Π±ΡƒΠΆΠ΄Π΅Π½Π½ΠΎΠ³ΠΎ состояния Π°ΠΊΡ†Π΅ΠΏΡ‚ΠΎΡ€Π° увСличиваСтся Π½Π° ΠΏΠΎΡ€ΡΠ΄ΠΊΠΈ ΠΏΠΎ ΡΡ€Π°Π²Π½Π΅Π½ΠΈΡŽ с Π΅ΡΡ‚СствСнным Π²Ρ€Π΅ΠΌΠ΅Π½Π΅ΠΌ ΠΆΠΈΠ·Π½ΠΈ флуорСсцСнции [12].

Π Π―Π•Π’-ΠΏΠ°Ρ€Ρ‹ Π½Π° ΠΎΡΠ½ΠΎΠ²Π΅ тСрбия Π² ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠ΅ с Π³Π΅Π½Π΅Ρ‚ичСски ΠΊΠΎΠ΄ΠΈΡ€ΡƒΠ΅ΠΌΡ‹ΠΌ Ρ‚Π΅Ρ€Π±ΠΈΠΉ-ΡΠ²ΡΠ·Ρ‹Π²Π°ΡŽΡ‰ΠΈΠΌ ΠΏΠ΅ΠΏΡ‚ΠΈΠ΄ΠΎΠΌ ΠΈ ΠΊΡ€Π°ΡΠ½Ρ‹ΠΌ флуорСсцСнтным Π±Π΅Π»ΠΊΠΎΠΌ ΠΈΠΌΠ΅ΡŽΡ‚ большоС ΠΏΠ΅Ρ€Π΅ΠΊΡ€Ρ‹Π²Π°Π½ΠΈΠ΅ спСктра флуорСсцСнции тСрбия ΠΈ Π²ΠΎΠ·Π±ΡƒΠΆΠ΄Π΅Π½ΠΈΡ красных флуорСсцСнтных Π±Π΅Π»ΠΊΠΎΠ², Ρ‡Ρ‚ΠΎ создаСт условия для эффСктивного пСрСноса энСргии. ИспользованиС спСктроскопии с Π²Ρ€Π΅ΠΌΠ΅Π½Π½ΠΎΠΉ Π·Π°Π΄Π΅Ρ€ΠΆΠΊΠΎΠΉ ΠΏΠΎΠ·Π²ΠΎΠ»ΠΈΡ‚ ΠΈΠ·Π±Π΅ΠΆΠ°Ρ‚ΡŒ рСгистрации ΠΊΠΎΡ€ΠΎΡ‚ΠΊΠΎΠΆΠΈΠ²ΡƒΡ‰Π΅Π³ΠΎ Ρ„ΠΎΠ½ΠΎΠ²ΠΎΠ³ΠΎ сигнала ΠΊΠ»Π΅Ρ‚ΠΎΠΊ, Ρ‡Ρ‚ΠΎ Π²Π°ΠΆΠ½ΠΎ Π² ΡƒΡΠ»ΠΎΠ²ΠΈΡΡ… Π½ΠΈΠ·ΠΊΠΎΠΉ интСнсивности сигнала Π Π«Π•Π’-ΠΏΠ°Ρ€Ρ‹.

ЦСль Ρ€Π°Π±ΠΎΡ‚Ρ‹: ΠΏΠΎΠ»ΡƒΡ‡Π΅Π½ΠΈΠ΅ гСнСтичСски ΠΊΠΎΠ΄ΠΈΡ€ΡƒΠ΅ΠΌΡ‹Ρ… Π ΠšΠ•Π’-сСнсоров каспазы-3 Π½Π° ΠΎΡΠ½ΠΎΠ²Π΅ Ρ‚Π΅Ρ€Π±ΠΈΠΉ-ΡΠ²ΡΠ·Ρ‹Π²Π°ΡŽΡ‰Π΅Π³ΠΎ ΠΏΠ΅ΠΏΡ‚ΠΈΠ΄Π° ΠΈ ΠΊΡ€Π°ΡΠ½Ρ‹Ρ… флуорСсцСнтных Π±Π΅Π»ΠΊΠΎΠ² Π‘Π·ΠšΠ΅Ρ12 ΠΈ Π’Π°§ КРР.

Π²Ρ‹Π²ΠΎΠ΄Ρ‹.

1. Π’ΠΏΠ΅Ρ€Π²Ρ‹Π΅ ΠΏΠΎΠ»ΡƒΡ‡Π΅Π½Ρ‹ гСнСтичСски ΠΊΠΎΠ΄ΠΈΡ€ΡƒΠ΅ΠΌΡ‹Π΅ Π ΠšΠ•Π’-ΠΏΠ°Ρ€Ρ‹ Π½Π° ΠΎΡΠ½ΠΎΠ²Π΅ флуорСсцСнтных Π±Π΅Π»ΠΊΠΎΠ² Π’ΡΠšΠ΅Ρ12 ΠΈ Π’Π°§ КРР, субстрата каспазы-3 ΠΈ Ρ‚Π΅Ρ€Π±ΠΈΠΉ-ΡΠ²ΡΠ·Ρ‹Π²Π°ΡŽΡ‰Π΅Π³ΠΎ ΠΏΠ΅ΠΏΡ‚ΠΈΠ΄Π°: Π’Π¬3±Π‘П-Π’Π•Π£Π’-Оз11Сс12 ΠΈ Tb3±CП-DEVD-TagRFP.

2. ΠŸΡ€ΠΎΠ΄Π΅ΠΌΠΎΠ½ΡΡ‚Ρ€ΠΈΡ€ΠΎΠ²Π°Π½ΠΎ Π½Π°Π»ΠΈΡ‡ΠΈΠ΅ Π΄Π²ΡƒΡ… процСссов ΠΈΠ½Π΄ΡƒΠΊΡ‚ΠΈΠ²Π½ΠΎ-рСзонансного пСрСноса энСргии Π² ΠΎΠ±Π΅ΠΈΡ… Π Π«Π•Π’-ΠΏΠ°Ρ€Π°Ρ…: ΠΎΡ‚ Ρ‚Π΅Ρ€Π±ΠΈΠΉ-ΡΠ²ΡΠ·Ρ‹Π²Π°ΡŽΡ‰Π΅Π³ΠΎ ΠΏΠ΅ΠΏΡ‚ΠΈΠ΄Π° ΠΊ ΠΈΠΎΠ½Ρƒ Π’Π¬3+, ΠΎΡ‚ ΠΈΠΎΠ½Π° Π’Π¬3+ ΠΊ Ρ…Ρ€ΠΎΠΌΠΎΡ„ΠΎΡ€Π°ΠΌ флуорСсцСнтных Π±Π΅Π»ΠΊΠΎΠ².

3. ΠžΠΏΡ€Π΅Π΄Π΅Π»Π΅Π½Π° ΡΡ„Ρ„Π΅ΠΊΡ‚ΠΈΠ²Π½ΠΎΡΡ‚ΡŒ пСрСноса энСргии ΠΎΡ‚ ΠΈΠΎΠ½Π° Π’Π¬3+ ΠΊ Ρ…Ρ€ΠΎΠΌΠΎΡ„ΠΎΡ€Π°ΠΌ флуорСсцСнтных Π±Π΅Π»ΠΊΠΎΠ², для Π±Π΅Π»ΠΊΠ° Π’Π¬3±Π‘П-Π’Π•Π£Π’-Π’Π·11Сс12, ΠΎΠ½Π° составила 28%, для Π±Π΅Π»ΠΊΠ° Π’Π¬3±Π‘П-ΠžΠ•Π£Π‘-Π’Π°§ Π‘^Π  — 35%.

4. Показано, Ρ‡Ρ‚ΠΎ Π΄ΠΎΠ±Π°Π²Π»Π΅Π½ΠΈΠ΅ Π«Π°Π‘1 Π² Ρ€Π°ΡΡ‚Π²ΠΎΡ€ с Π³ΠΈΠ±Ρ€ΠΈΠ΄Π½Ρ‹ΠΌΠΈ Π±Π΅Π»ΠΊΠ°ΠΌΠΈ ΠΏΡ€ΠΈΠ²ΠΎΠ΄ΠΈΡ‚ ΠΊ Π΄Π΅Π·Π°ΠΊΡ‚ΠΈΠ²Π°Ρ†ΠΈΠΈ Π²ΠΎΠ·Π±ΡƒΠΆΠ΄Π΅Π½Π½ΠΎΠ³ΠΎ состояния Π’Π¬3+, ΠΈ, ΠΊΠ°ΠΊ слСдствиС, ΠΊ ΡΠ½ΠΈΠΆΠ΅Π½ΠΈΡŽ интСнсивности флуорСсцСнции ΠΈ Π²Ρ€Π΅ΠΌΠ΅Π½ΠΈ ΠΆΠΈΠ·Π½ΠΈ Π’Π¬3+ Π² ΠΎΠ±Π΅ΠΈΡ… Π¨Π•Π’-ΠΏΠ°Ρ€Π°Ρ…. Π”Π°Π½Π½Ρ‹ΠΉ эффСкт ΠΏΡ€Π΅Π΄ΠΏΠΎΠ»ΠΎΠΆΠΈΡ‚Π΅Π»ΡŒΠ½ΠΎ связан с Π΄ΠΈΠ½Π°ΠΌΠΈΡ‡Π΅ΡΠΊΠΈΠΌ Ρ‚ΡƒΡˆΠ΅Π½ΠΈΠ΅ΠΌ флуорСсСцСнции тСрбия ΠΈΠΎΠ½Π°ΠΌΠΈ Π‘Π“.

5. Показано, Ρ‡Ρ‚ΠΎ Π΄ΠΈΠΌΠ΅Ρ€Π½Ρ‹ΠΉ субстрат Π’Π¬3±Π‘П-Π‘Π•Π£Π‘-Π’Π°§ КРР подвСргаСтся Π³ΠΈΠ΄Ρ€ΠΎΠ»ΠΈΠ·Ρƒ каспазой-3, Ρ‡Ρ‚ΠΎ ΠΏΠΎΠ΄Ρ‚Π²Π΅Ρ€ΠΆΠ΄Π΅Π½ΠΎ ΠΌΠ΅Ρ‚ΠΎΠ΄Π°ΠΌΠΈ фосфорСсцСнтной спСктроскопии ΠΈ ΡΠ»Π΅ΠΊΡ‚Ρ€ΠΎΡ„ΠΎΡ€Π΅Π·Π°.

6. Π’ ΡΠ»ΡƒΡ‡Π°Π΅ Ρ‚Π΅Ρ‚Ρ€Π°ΠΌΠ΅Ρ€Π½ΠΎΠ³ΠΎ Π±Π΅Π»ΠΊΠ° Π’Π¬3±Π‘П-Π’Π•Π£Π’-Π‘511Сс12 Π½Π°Π±Π»ΡŽΠ΄Π°Π΅Ρ‚ΡΡ отсутствиС Π³ΠΈΠ΄Ρ€ΠΎΠ»ΠΈΠ·Π° каспазой-3 Π·Π° ΠΈΡΡΠ»Π΅Π΄ΡƒΠ΅ΠΌΡ‹ΠΉ ΠΏΡ€ΠΎΠΌΠ΅ΠΆΡƒΡ‚ΠΎΠΊ Π²Ρ€Π΅ΠΌΠ΅Π½ΠΈ, Ρ‡Ρ‚ΠΎ ΠΏΡ€Π΅Π΄ΠΏΠΎΠ»ΠΎΠΆΠΈΡ‚Π΅Π»ΡŒΠ½ΠΎ ΠΎΠ±ΡŠΡΡΠ½ΡΠ΅Ρ‚ΡΡ влияниСм стСричСских Ρ„Π°ΠΊΡ‚ΠΎΡ€ΠΎΠ² Π½Π° Π΄ΠΎΡΡ‚ΡƒΠΏ каспазы-3 ΠΊ ΡΡƒΠ±ΡΡ‚Ρ€Π°Ρ‚Ρƒ.

Π—Π°ΠΊΠ»ΡŽΡ‡Π΅Π½ΠΈΠ΅

.

НаиболСС подходящим субстратом для in vivo опрСдСлСния активности каспазы-3 i i ΡΠ²Π»ΡΠ΅Ρ‚ся субстрат ВЬБП-DE VD-TagRFP. Данная FRET-napa Π²ΠΏΠ΅Ρ€Π²Ρ‹Π΅ ΠΏΠΎΠ»ΡƒΡ‡Π΅Π½Π° Π½Π° ΠΎΡΠ½ΠΎΠ²Π΅ тСрбия Π² ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠ΅ с Ρ‚Π΅Ρ€Π±ΠΈΠΉ-ΡΠ²ΡΠ·Ρ‹Π²Π°ΡŽΠ³Ρ†ΠΈΠΌ ΠΏΠ΅ΠΏΡ‚ΠΈΠ΄ΠΎΠΌ ΠΈ ΠΊΡ€Π°ΡΠ½ΠΎΠ³ΠΎ флуорСсцСнтного Π±Π΅Π»ΠΊΠ° TagRFP ΠΈ ΡΠ²Π»ΡΠ΅Ρ‚ся ΠΏΠΎΠ»Π½ΠΎΡΡ‚ΡŒΡŽ гСнСтичСски ΠΊΠΎΠ΄ΠΈΡ€ΡƒΠ΅ΠΌΠΎΠΉ. Π­Ρ‚ΠΎ Π²Π°ΠΆΠ½ΠΎ ΠΏΡ€ΠΈ использовании сСнсора Π² ΠΆΠΈΠ²Ρ‹Ρ… ΠΈ ΠΏΠΎΡΡ‚оянно дСлящихся ΠΊΠ»Π΅Ρ‚ΠΊΠ°Ρ…, Π² ΡΠ²ΡΠ·ΠΈ с ΠΎΡ‚сутствиСм нСобходимости внСшнСй ΠΈΠ½ΡŠΠ΅ΠΊΡ†ΠΈΠΈ. Π‘ ΠΏΠΎΠΌΠΎΡ‰ΡŒΡŽ Π΄Π°Π½Π½ΠΎΠ³ΠΎ FRET-сСнсора ΠΌΠΎΠΆΠ½ΠΎ эффСктивно ΡΠ½ΠΈΠ·ΠΈΡ‚ΡŒ ΠΊΠΎΡ€ΠΎΡ‚ΠΊΠΎΠΆΠΈΠ²ΡƒΡ‰ΠΈΠΉ Ρ„ΠΎΠ½ΠΎΠ²Ρ‹ΠΉ сигнал Π² ΠΆΠΈΠ²Ρ‹Ρ… ΠΊΠ»Π΅Ρ‚ΠΊΠ°Ρ…, Ρ‡Ρ‚ΠΎ Π²Π°ΠΆΠ½ΠΎ Π² ΡƒΡΠ»ΠΎΠ²ΠΈΡΡ… рСгистрации Π½ΠΈΠ·ΠΊΠΎΠΉ интСнсивности сигнала.

ΠŸΠΎΠΊΠ°Π·Π°Ρ‚ΡŒ вСсь тСкст

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