WO2016095458A1

WO2016095458A1 - Homogenizer for separating subcellular fractions

Info

Publication number: WO2016095458A1
Application number: PCT/CN2015/081070
Authority: WO
Inventors: 何向锋; 强福林; 沈康; 施文; 王健
Original assignee: 南通市肿瘤医院
Priority date: 2014-12-16
Filing date: 2015-06-09
Publication date: 2016-06-23
Also published as: CN104498350A; CN104498350B

Abstract

Disclosed is a homogenizer for separating subcellular fractions, which can be used for preparing homogenate containing various subcellular fractions from cells or tissue blocks. The homogenizer comprises a homogenization tube. The homogenization tube comprises a tube wall and a tube core located in the tube wall, the top part of the tube wall is closed, a tube cover is covered at the bottom of the tube wall, the tube core comprises a stator sleeve and a rotor located in the sleeve, a rotor joint is disposed in the tube cover, and the rotor is connected to the rotor joint. The homogenizer further comprises a motor located outside the homogenization tube. The motor provides rotation power for the rotor by using the rotor joint. The homogenization tube can be designed into a tissue homogenization tube or a cell homogenization tube as required, to facilitate change use. An automatic system with a special tube core is used in the present invention, a special application program is defined by a microprocessor to optimize an operation procedure, so that a sample preparation process is standardized and programmed, the influence of artificial factors can be reduced to obtain homogenate with a subcellular structure, which is used for separating the subcellular structure through density gradient centrifugation and differential speed centrifugation.

Description

A homogenizer for separating subcellular components

Technical field

The present invention relates to the separation of subcellular structures, and in particular to a homogenizer for separating subcellular components.

Background technique

Cells are composed of various subcellular structures, including nucleus, mitochondria, lysosomes, autophagosomes, endoplasmic reticulum, and Golgi. To observe these subcellular structures or perform biochemical functional analysis, various subcellular groups need to be isolated and purified. Centrifugal technology is the basic means to achieve this goal. Prior to centrifugation, a cell homogenate or tissue homogenate needs to be prepared. The homogenization is carried out by placing the cells or tissues in a homogenizer under low temperature conditions and adding an isotonic homogenate medium (ie 0.25 mol/L sucrose-0.003 mol/L calcium chloride solution) to grind the cells or tissues mechanically. It is ground to a mixture containing various subcellular components and inclusions.

There are various ways of homogenization: manual homogenization, machine homogenization, ultrasonic homogenization, repeated freezing and thawing, etc., which can soften the homogenization of animal and plant tissues or cells to obtain the tissue homogenate or cell homogenate required for the experiment. .

Manual homogenization is to homogenize the tissue fragments or cells together with the homogenization medium or physiological saline into the glass homogenate tube. The left hand is held in the homogenization tube and the lower end is inserted into the vessel containing the ice water mixture. The right hand will be the mast vertical. Insert into the cannula, rotate it up and down for several dozen times (6-8 minutes), and grind it thoroughly to homogenize the tissue. Manual homogenization using a glass homogenizer is laborious, time consuming, and not uniform enough, and increases the probability of contamination. The homogenization of the machine is based on the principle of grinding, using the machine as the power, driving the homogenizing rod to move in the homogenizer, and softening and homogenizing the animal and plant tissues to obtain the cell homogenate or tissue homogenate required for the experiment. Although the machine homogenization has liberated manpower, it is still open operation and easy to pollute. Ultrasonic pulverization is the use of an ultrasonic cell disruptor to break up cells, easily damage the organelles, and affect the activity of certain enzymes. The cultured or isolated cells can be homogenized by the above method, or can be repeatedly frozen and lyophilized about 3 times (ie, let the cells add appropriate amount of hypotonic solution or double distilled water to freeze in a low temperature refrigerator, dissolve, freeze, and dissolve again. , repeated about 3 times), but some enzyme activities will be affected, and the organelles are not completely released.

Summary of the invention

The object of the present invention is to provide a tissue or cell homogenizer for separating subcellular structures, which is simple, rapid and efficient for preparing tissue or cell homogenate.

The technical solution of the present invention is:

A homogenizer for separating subcellular components, separating a cell or tissue block into a subcellular structure, the homogenizer comprising a homogenate tube comprising a tube wall and a tube located within the tube wall a core, the top of the tube wall is closed, and the bottom is covered with a tube cover, the wick includes a stator sleeve and a rotor located in the sleeve, the tube cover is provided with a rotor joint, and the rotor is connected with the rotor joint, the separation The machine also includes a motor external to the homogenizing tube that provides rotational power to the rotor through the rotor joint.

A preferred solution is that the inside of the die of the tissue homogenizer is divided into an Archimedes spiral tube region and a homogenized grinding region from top to bottom, and the Archimedes spiral tube region is provided with a screw rod and homogenized. The grinding zone is provided with a grinding rod, the auger and the grinding rod are connected to form a rotor, and the bottom of the grinding rod is connected with the rotor joint, and the inner wall of the stator sleeve of the homogenizing grinding zone is provided with an abrasive layer corresponding to the position of the grinding rod. The stator sleeve base is provided with a plurality of evenly distributed holes. Further preferably, the hole has a diameter of 2.0 to 3.0 mm.

Another preferred solution is that the inner core of the cell homogenate tube is divided into an Archimedes spiral tube region, a homogenized grinding region and a negative pressure rotary cutting region from top to bottom, and the Archimedes spiral tube region A screw rod is arranged therein, a grinding rod is arranged in the homogenizing grinding area, and a rotary cutting head is arranged in the negative pressure rotary cutting area, and the spiral rod, the grinding rod and the rotary cutting head are sequentially connected to form a rotor, and the bottom of the rotary cutting head is connected with the rotor Connector. The inner wall of the stator sleeve of the homogenized grinding zone is provided with an abrasive layer corresponding to the position of the grinding rod. The stator sleeve of the negative pressure rotary cutting zone is provided with a plurality of evenly distributed holes corresponding to the portion of the rotary cutter head. Further preferably, the hole has a diameter of 0.8 to 1.2 mm.

For the above two options:

Preferably, the rotor is sealed by a joint of the rotor joint and the tube cover.

Preferably, the outer portion of the homogenizing tube is further provided with a protective heat preservation cover.

Further preferably, the protective heat insulating cover is provided with a temperature probe and a refrigeration compressor, wherein the temperature probe and the refrigeration compressor are respectively connected to a microprocessor, and the refrigeration compressor is further connected with a circulation fan; the microprocessor Also connected to the motor and a touch screen.

The advantages of the invention are:

1. The homogenizer for separating subcellular structures provided by the invention is combined with a tissue homogenate tube or a cell homogenate tube, a special die is built in, and an automatic system is formed, and the microprocessor defines a special application (time, speed, Direction) Optimize the operating procedures, conveniently process the samples, standardize and program the sample preparation process, reduce the influence of human factors, and change the operator dependence to non-operator dependencies.

2. The homogenizer of the invention can select different homogenizing tubes according to requirements, and when the sample is a tissue block, the tissue with the Archimedes spiral tube region, the homogenized grinding region and the negative pressure rotary cutting region can be used. In the slurry tube, when the sample is a single cell suspension, the cell homogenate tube with the Archimedes spiral tube region and the homogenized grinding region can be directly used, and the switching is flexible. When using the tissue homogenization tube, the tissue sample required can be simply prepared into a tissue block of several millimeters before separation. The rotary cutter in the tissue homogenization tube can be automatically cut into tiny pieces of 1.0 mm3. Further, a subcellular structure homogenate is prepared through the homogenized grinding zone, and the subcellular structure can be separated by density gradient centrifugation and differential centrifugation.

3. The present invention can also be used to prepare tissue protein homogenates by setting a specific procedure to control speed, time and direction.

4. The invention has a protective heat preservation cover, controls the temperature in the protective heat preservation cover to be 0-4 ° C, protects the organelle and protein function, and provides a sealed aseptic working environment to ensure the safety of the user.

5. The invention is gentle in operation, simple, fast and efficient.

DRAWINGS

The present invention is further described below in conjunction with the accompanying drawings and embodiments:

1 is a structural principle of a homogenizer for separating a subcellular structure according to the present invention;

2 is a schematic structural view of a tissue homogenate tube according to the present invention;

Figure 3 is an exploded view of the tissue homogenate tube of Figure 2;

Figure 4 is a schematic view showing the structure of a cell homogenate tube according to the present invention;

Figure 5 is an exploded view of the cell homogenate tube shown in Figure 4;

Figure 6 is a schematic diagram of the temperature probe of the present invention.

detailed description

As shown in FIG. 1 , the homogenizer disclosed in the present invention for separating a tissue block or a cell homogenate into a subcellular structure includes a homogenizing tube, and a motor is arranged under the homogenizing tube, and the motor speed is adjustable, and can be reversed. Turn, it can be operated according to the set program (time, speed, direction). The outside of the homogenizing tube is further provided with a protective heat preservation cover, wherein the protection heat preservation cover is provided with a temperature probe, a refrigeration compressor and a circulation fan, and the temperature probe, the refrigeration compressor and the circulation fan are respectively connected with a microprocessor to realize The temperature inside the protective heat shield is automatically controlled by 0-4 °C to protect the organelles and protein function. The microprocessor is also connected to the motor to control the rotation speed and direction of the motor. The microprocessor is also connected with a touch screen for inputting operating parameters and displaying the running process.

Example 1

As shown in Figures 2 and 3, a tissue homogenate tube is provided which can be used when the sample is a tissue block, the tissue homogenate tube comprising the tube wall 1 and a tube located within the tube wall Core 2, the top of the tube wall is closed, and the bottom is covered with a tube cover 3, the core 2 includes a stator sleeve 4 and a rotor 5 located in the sleeve 4, the tube cover 3 is provided with a rotor joint, the rotor and The rotor joint is connected, the rotor is sealed by the joint of the rotor joint and the tube cover, and the motor below the homogenizing tube provides rotational power to the rotor through the rotor joint, and the stator sleeve 4 and the tube wall are fixed to the motor through the bayonet.

The die 2 is divided into an Archimedes spiral tube region, a homogenized grinding region and a negative pressure rotary cutting region from top to bottom, and the lower portion of the stator sleeve of the die is provided with a plurality of uniformly distributed diameters corresponding to the negative pressure rotary cutting region. It is a hole 6 of 0.8-1.2 mm. a screw rod 51 is arranged in the Archimedes spiral tube zone, a grinding rod 52 is arranged in the homogenized grinding zone, and a rotary cutter head 53 is arranged in the negative pressure rotary cutting zone, the screw rod 51, the grinding rod 52 and the rotary cutter The head 53 is sequentially connected to form a rotor 5, and the bottom of the rotary cutter head 53 is connected to the rotor joint.

Among them: Archimedes spiral tube area: the spiral rod structure of the rotor and the stator sleeve together form an Archimedes spiral tube, which uses the spiral wheel in the casing cylinder to rise and lift the liquid. The working principle is that when the inner spiral rotates, The screw rotates on its own axis on the one hand and on the other hand it rolls along the inner surface of the bushing, thus forming a sealed chamber of liquid. Every revolution of the screw, the water in the sealed chamber advances by a pitch. As the screw rotates continuously, the water is pressed from one sealed chamber to the other, and finally the liquid is extruded from the top of the sleeve. A negative pressure is formed at the bottom, causing suction.

Negative pressure rotary cutting zone: the suction formed by the negative pressure makes the tissue block close to the casing wall and sinks into the hole. The high-speed rotating rotary cutting head cuts the tissue trapped in the cavity to form a tiny tissue of 1.0mm3 size. Piece.

Homogenized grinding zone: The micro-structured block is lifted by the liquid under the suction of the negative pressure, passes through the homogenized grinding zone, the Teflon homogenate working surface on the polished rod and the Teflon abrasive layer on the inner wall of the die The ground is broken into subcellular structure homogenates.

The liquid in the homogenizing tube carries tiny tissue blocks continuously from the small holes in the bottom of the stator casing, and passes through the negative pressure rotary cutting zone, the homogenized grinding zone and the Archimedes spiral pipe zone, and flows out from the top of the casing, so that the process is repeated. The tissue is ground into a subcellular homogenate. The tissue homogenization process is designed with a reversal process. When the rotor is reversed, the liquid flow in the die is reversed, which can prevent the tissue fragments from blocking the holes in the negative pressure rotary cutting zone.

The sample quality range of the present invention is 20-4000 mg; the rotor speed: 50-1000 rpm, which can be rotated in the forward and reverse directions; time: 1-600 seconds; temperature: controllable temperature is 0-4 ° C; application: according to different tissues Block characteristics and homogenization requirements provide optimized operating procedures and homogenization procedures; specimen processing: a sterile, confined space within the tissue homogenate tube.

The temperature probe, the refrigeration compressor and the circulation fan of the present invention jointly achieve the purpose of refrigeration.

As shown in FIG. 6, the temperature probe of the temperature probe of the present invention employs a digital temperature sensor. This sensor integrates temperature sensing, signal conversion, A/D conversion and other functions into one chip. Its internal structure is shown in Figure 6. The chip converts the temperature into an electrical signal that first enters the weak signal amplifier for amplification; then enters a 14-bit A/D converter; finally, the digital signal is output via a two-wire serial digital interface. This sensor has a calibration register that automatically calibrates the signal from the sensor during the measurement.

Example 2

As shown in Figures 4 and 5, a cell homogenate tube is provided which can be used when the sample is a single cell suspension. The cell homogenate tube comprises a tube wall 1' and a tube 2' located within the tube wall, the tube wall being closed at the top, the bottom portion being covered with a tube cover 3', the tube 2' comprising a stator sleeve 4' and a rotor 5' located in the sleeve 4', the tube cover 3' is provided with a rotor joint, the rotor is connected to the rotor joint, the rotor is sealed by the joint of the rotor joint and the tube cover, and the motor below the separation tube passes through the rotor The joint provides rotational power to the rotor, and the stator sleeve and the tube wall are secured to the motor by a bayonet.

The inner 2' of the die is divided into an Archimedes spiral tube region and a homogenized grinding region from top to bottom, and the stator sleeve base of the die is provided with a plurality of uniformly distributed holes 6' having a diameter of 2-3 mm. A screw rod 51' is arranged in the Archimedes spiral tube zone, and a grinding rod 52' is arranged in the homogenized grinding zone. The screw rod 51' and the grinding rod 52' are connected to form a rotor 5', and the bottom is connected to the rotor joint. The functions of the Archimedes spiral zone and the homogenized grinding zone were the same as in Example 1.

The sample volume range of the cell homogenate tube: 20-40 ml; the rotor speed: 50-1000 rpm, which can be rotated in the forward and reverse directions; time: 1-600 seconds; temperature: the temperature can be controlled at 0-4 ° C; application: according to different Separation of subcellular structures requires optimized protocols and homogenization procedures; specimen processing: sterile, confined spaces within the homogenized tubes.

The temperature control portion of this embodiment is the same as that of the first embodiment.

The above embodiments are merely illustrative of the technical concept and the features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the present invention and to implement the present invention, and the scope of the present invention is not limited thereto. Modifications made in accordance with the spirit of the main technical solutions of the present invention are intended to be included within the scope of the present invention.

Claims

A homogenizer for separating subcellular components, the cells or tissue blocks being prepared into a cell homogenate or tissue homogenate comprising various subcellular components, the homogenizer comprising a homogenate tube, a homogenate tube The invention comprises a tissue homogenizing tube and a cell homogenizing tube, wherein the homogenizing tube comprises a tube wall and a tube located inside the tube wall, the tube wall is closed at the top, and the bottom portion is covered with a tube cover, and the tube core comprises a stator sleeve and a rotor located in the sleeve, the tube cover is provided with a rotor joint, the rotor is connected with the rotor joint, and the homogenizer further comprises a motor located outside the homogenizing tube, the motor is a rotor through the rotor joint Provides rotary power.
The cell homogenizer for separating subcellular components according to claim 1, wherein the inside of the die is divided into an Archimedes spiral tube region and a homogenized polishing region from top to bottom, A screw rod is arranged in the spiral zone of the Archimedes, and a grinding rod is arranged in the homogenized grinding zone, and the screw rod and the grinding rod are connected to form a rotor, and the bottom of the grinding rod is connected with the rotor joint, and the inner wall of the stator sleeve of the homogenized grinding area corresponds to The area of the grinding rod is provided with an abrasive layer.
The tissue homogenizer for separating sub-cellular components according to claim 1, wherein the inner core is divided into an Archimedes spiral tube region and a homogenized grinding region and a negative pressure from top to bottom. a rotary cutting section, wherein the Archimedes spiral tube area is provided with a screw rod, the grinding block is provided with a grinding rod, and the negative pressure rotary cutting area is provided with a rotary cutting head. The auger, the grinding rod and the rotary cutter head are sequentially connected to form a rotor, and the bottom of the rotary cutter head is connected with the rotor joint, and the inner wall of the stator sleeve of the homogenization grinding zone is provided with an abrasive layer corresponding to the position of the grinding rod.
A cell homogenizer for separating subcellular components according to claim 2, wherein said stator cannula base is provided with a plurality of uniformly distributed holes.

The tissue homogenizer for separating sub-cellular components according to claim 1, wherein the inner core is divided into an Archimedes spiral tube region and a homogenized grinding region and a negative pressure from top to bottom. a rotary cutting section, wherein the Archimedes spiral tube area is provided with a screw rod, the grinding block is provided with a grinding rod, and the negative pressure rotary cutting area is provided with a rotary cutting head. The auger, the grinding rod and the rotary cutter head are sequentially connected to form a rotor, and the bottom of the rotary cutter head is connected with the rotor joint, and the inner wall of the stator sleeve of the homogenization grinding zone is provided with an abrasive layer corresponding to the position of the grinding rod.
The homogenizer for separating subcellular components according to claim 4, wherein the pores have a diameter of 2.0 to 3.0 mm.
The tissue homogenizer for separating sub-cellular components according to claim 3, wherein the stator sleeve is provided with a plurality of uniformly distributed holes corresponding to portions of the negative pressure rotary cutting zone.
A tissue homogenizer for separating subcellular components according to claim 6, wherein the pores have a diameter of 0.8 to 1.2 mm.
A homogenizer for separating subcellular components according to claim 1, wherein said rotor is sealed by a joint of a rotor joint and a tube cap.
The homogenizer for separating sub-cellular components according to claim 2 or 3, characterized in that the outer portion of the homogenizing tube is further provided with a protective heat shield.
The homogenizer for separating sub-cellular components according to claim 2 or 3, wherein the protective heat insulating cover is provided with a temperature probe and a refrigeration compressor, and the temperature probe and the refrigeration compressor are respectively A microprocessor is connected, and the refrigeration compressor is further connected with a circulation fan; the microprocessor is also connected to the motor and a touch screen.